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Upon encountering macrophage colony-stimulating factor (M-CSF), human monocytes undergo a series of cellular signaling events leading to an increase in Akt activity. However, the regulation of these events is not completely understood. Because the inositol 5'-phosphatase SHIP-1 is an important regulator of intracellular levels of phosphatidylinositol 3,4,5-trisphosphate, an important second messenger necessary for Akt activation, we hypothesized that SHIP-1 was involved in the regulation of M-CSF receptor (M-CSF-R)-induced Akt activation. In the human monocytic cell line, THP-1, SHIP-1 became tyrosine-phosphorylated following M-CSF activation in a Src family kinase-dependent manner. Transfection of 3T3-Fms cells, which express the human MCSF-R, with wild-type SHIP-1 showed that SHIP-1 was necessary for the negative regulation of M-CSF-induced Akt activation. In THP-1 cells, SHIP-1 bound Lyn, independent of the kinase activity of Lyn, following M-CSF activation. Utilizing a glutathione S-transferase fusion protein, we found that SHIP-1 bound to Lyn via the SHIP-1 Src homology 2 domain. Furthermore, transfection of THP-1 cells with a wild-type SHIP-1 construct reduced NF-κB-dependent transcriptional activation of a reporter gene, whereas a SHIP-1 Src homology 2 domain construct resulted in an increase in NF-κB activation. Additionally, in 3T3-Fms cells, Lyn enhanced the ability of SHIP-1 to regulate Akt activation by stabilizing SHIP-1 at the cellular membrane. Finally, macrophages isolated from both SHIP-1- and Lyn-deficient mice exhibited enhanced Akt phosphorylation following M-CSF stimulation. These data provide the first evidence of the involvement of both SHIP-1 and Lyn in the negative regulation of M-CSF-R-induced Akt activation. [ABSTRACT FROM AUTHOR]

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of dense stroma that is enriched in hyaluronan (HA), with increased HA levels associated with more aggressive disease. Increased levels of the HA-degrading enzymes hyaluronidases (HYALs) are also associated with tumor progression. In this study, we evaluate the regulation of HYALs in PDAC. Methods: Using siRNA and small molecule inhibitors, we evaluated the regulation of HYALs using quantitative real-time PCR (qRT-PCR), Western blot analysis, and ELISA. The binding of BRD2 protein on the HYAL1 promoter was evaluated by chromatin immunoprecipitation (ChIP) assay. Proliferation was evaluated by WST-1 assay. Mice with xenograft tumors were treated with BET inhibitors. The expression of HYALs in tumors was analyzed by immunohistochemistry and by qRT-PCR. Results: We show that HYAL1, HYAL2, and HYAL3 are expressed in PDAC tumors and in PDAC and pancreatic stellate cell lines. We demonstrate that inhibitors targeting bromodomain and extra-terminal domain (BET) proteins, which are readers of histone acetylation marks, primarily decrease HYAL1 expression. We show that the BET family protein BRD2 regulates HYAL1 expression by binding to its promoter region and that HYAL1 downregulation decreases proliferation and enhances apoptosis of PDAC and stellate cell lines. Notably, BET inhibitors decrease the levels of HYAL1 expression in vivo without affecting the levels of HYAL2 or HYAL3. Conclusions: Our results demonstrate the pro-tumorigenic role of HYAL1 and identify the role of BRD2 in the regulation of HYAL1 in PDAC. Overall, these data enhance our understanding of the role and regulation of HYAL1 and provide the rationale for targeting HYAL1 in PDAC. [ABSTRACT FROM AUTHOR]

Overactivation of immune responses is a hallmark of autoimmune disease pathogenesis. This includes the heightened production of inflammatory cytokines such as Tumor Necrosis Factor α (TNFα), and the secretion of autoantibodies such as isotypes of rheumatoid factor (RF) and anticitrullinated protein antibody (ACPA). Fcγ receptors (FcγR) expressed on the surface of myeloid cells bind Immunoglobulin G (IgG) immune complexes. Recognition of autoantigen-antibody complexes by FcγR induces an inflammatory phenotype that results in tissue damage and further escalation of the inflammatory response. Bromodomain and extra-terminal protein (BET) inhibition is associated with reduced immune responses, making the BET family a potential therapeutic target for autoimmune diseases such as rheumatoid arthritis (RA). In this paper, we examined the BET inhibitor PLX51107 and its effect on regulating FcγR expression and function in RA. PLX51107 significantly downregulated expression of FcγRIIa, FcγRIIb, FcγRIIIa, and the common γ-chain, FcϵR1-γ, in both healthy donor and RA patient monocytes. Consistent with this, PLX51107 treatment attenuated signaling events downstream of FcγR activation. This was accompanied by a significant decrease in phagocytosis and TNFα production. Finally, in a collagen-induced arthritis model, PLX51107-treatment reduced FcγR expression in vivo accompanied by a significant reduction in footpad swelling. These results suggest that BET inhibition is a novel therapeutic approach that requires further exploration as a treatment for patients with RA. [ABSTRACT FROM AUTHOR]

TLRs, a family of membrane-bound pattern recognition receptors found on innate immune cells, have been well studied in the context of cancer therapy. Activation of these receptors has been shown to induce inflammatory anticancer events, including differentiation and apoptosis, across a wide variety of malignancies. In contrast, intracellular pattern recognition receptors such as NOD-like receptors have been minimally studied. NOD2 is a member of the NOD-like receptor family that initiates inflammatory signaling in response to the bacterial motif muramyl dipeptide. In this study, we examined the influence of NOD2 in human acute myeloid leukemia (AML) cells, demonstrating that IFN-g treatment upregulated the expression of NOD2 signaling pathway members SLC15A3 and SLC15A4, downstream signaling kinase RIPK2, and the NOD2 receptor itself. This priming allowed for effective induction of caspase-1-dependent cell death upon treatment with muramyl tripeptide phosphatidylethanolamine (MTP-PE), a synthetic ligand for NOD2. Furthermore, the combination of MTP-PE and IFN-g on AML blasts generated an inflammatory cytokine profile and activated NK cells. In a murine model of AML, dual treatment with MTP-PE and IFN-g led to a significant increase in mature CD272 CD11b+ NK cells as well as a significant reduction in disease burden and extended survival. These results suggest that NOD2 activation, primed by IFN-g, may provide a novel therapeutic option for AML. [ABSTRACT FROM AUTHOR]

Monocytes and macrophages express FcγR that engage IgG immune complexes such as Ab-opsonized pathogens or cancer cells to destroy them by various mechanisms, including phagocytosis. FcγR-mediated phagocytosis is regulated by the concerted actions of activating FcγR and inhibitory receptors, such as FcγRIIb and SIRPα. In this study, we report that another ITIM-containing receptor, PECAM1/CD31, regulates FcγR function and is itself regulated by FcγR activation. First, quantitative RT-PCR and flow cytometry analyses revealed that human monocyte FcγR activation leads to a significant downregulation of CD31 expression, both at the message level and at surface expression, mainly mediated through FcγRIIa. Interestingly, the kinetics of downregulation between the two varied, with surface expression reducing earlier than the message. Experiments to analyze the mechanism behind this discrepancy revealed that the loss of surface expression was because of internalization, which depended predominantly on the PI3 kinase pathway and was independent of FcγR internalization. Finally, functional analyses showed that the downregulation of CD31 expression in monocytes by small interfering RNA enhanced FcγR-mediated phagocytic ability but have little effect on cytokine production. Together, these results suggest that CD31 acts as a checkpoint receptor that could be targeted to enhance FcγR functions in Ab-mediated therapies. [ABSTRACT FROM AUTHOR]

Background: Tumor-associated macrophages (TAM) are expanded and exhibit tumor-promoting properties within the tumor microenvironment. Current methods to study TAM have not been replicated across cancer types and often do not include exogenous growth factors from the tumor, a key factor in TAM differentiation in vivo. Methods: In this study, an in vitro method to generate monocyte- derived TAM using tumor- conditioned media (TCM) and a cytokine cocktail containing IL-4, IL-10, and M-CSF was utilized to study the phenotype, morphology, and function of TAM across multiple cancer types. TCM was generated from two breast cancer cell lines and an Epstein-Barr virus-positive lymphoma cell line. The properties of in vitro generated TAM were compared to in vitro generated M1 and M2- like macrophages and TAM isolated from patients with cancer. Results: TAM generated in this fashion displayed an increase in CD163/CD206 co-expression compared to M2- like macrophages (87 and 36%, respectively). TAM generated in vitro exhibited increased transcript levels of the functional markers IL-6, IL-10, CCL2, c-Myc, iNOS, and arginase compared to in vitro generated M2-like macrophages. Functionally, in vitro generated TAM inhibited the proliferation of T cells (47% decrease from M1-like macrophages) and the production of IFN-γ by natural killer cells was inhibited (44%) when co-cultured with TAM. Furthermore, in vitro generated TAM secreted soluble factors that promote the growth and survival of tumor cells. Conclusions: Limited access to patient TAM highlights the need for methods to generate TAM in vitro. Our data confirm that monocyte-derived TAM can be generated reliably using TCM plus the cytokine cocktail of IL-4, IL-10, and M-CSF. Given the ability of TAM to inhibit immune cell function, continued study of methods to deplete or deactivate TAM in the setting of cancer are warranted. [ABSTRACT FROM AUTHOR]

An inflammatory microenvironment has been shown to play an important role in the growth and metastasis of tumors. The NLRP3 inflammasome is a multi-protein complex of the innate immune system that is responsible for the production of the potent inflammatory cytokine IL-1β. Tumor- associated macrophages (TAM) are an expanded population of immune cells found in the tumor microenvironment that can promote the initiation and metastasis of tumor cells. Their presence has been correlated with disease burden, highlighting the therapeutic potential of targeting this population. However, to date clinically relevant pharmacologic strategies to target TAM remain elusive. Here, we show that in vitro generated TAM harbor NLRP3 inflammasome components and produce IL-1β. Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK), is in clinical use for the treatment of B- cell malignancies. We report that BTK is expressed by human in vitro generated TAM and murine macrophages and that it physically associates with the NLRP3 inflammasome. Furthermore, ibrutinib is able to inhibit BTK phosphorylation in TAM generated in vitro. Treatment of TAM with ibrutinib significantly impaired the ability of these cells to produce IL-1β. The present study provides evidence that BTK physically associates with the NLRP3 inflammasome and that inhibition of BTK with ibrutinib can impair the production of IL-1β by in vitro generated TAM. Thus, ibrutinib could potentially be of clinical use in abrogating inflammation-associated cancer progression and the immune-suppressive effects of myeloid cells within the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Activating mutations in BRAF are found in 50% of melanomas and although treatment with BRAF inhibitors (BRAFi) is effective, resistance often develops. We now show that recently discovered NRAS isoform 2 is up-regulated in the setting of BRAF inhibitor resistance in melanoma, in both cell lines and patient tumor tissues. When isoform 2 was overexpressed in BRAF mutant melanoma cell lines, melanoma cell proliferation and in vivo tumor growth were significantly increased in the presence of BRAFi treatment. shRNA-mediated knockdown of isoform 2 in BRAFi resistant cells restored sensitivity to BRAFi compared with controls. Signaling analysis indicated decreased mitogen-activated protein kinase (MAPK) pathway signaling and increased phosphoinositol-3-kinase (PI3K) pathway signaling in isoform 2 overexpressing cells compared with isoform 1 overexpressing cells. Immunoprecipitation of isoform 2 validated a binding affinity of this isoform to both PI3K and BRAF/RAF1. The addition of an AKT inhibitor to BRAFi treatment resulted in a partial restoration of BRAFi sensitivity in cells expressing high levels of isoform 2. NRAS isoform 2 may contribute to resistance to BRAFi by facilitating PI3K pathway activation. [ABSTRACT FROM AUTHOR]

Active Hexose Correlated Compound (AHCC) has been shown to have many immunostimulatory and anti-cancer activities in mice and in humans. As a natural product, AHCC has potential to create safer adjuvant therapies in cancer patients. Acute Myeloid Leukemia (AML) is the least curable and second-most common leukemia in adults. AML is especially terminal to those over 60 years old, where median survival is only 5 to 10 months, due to inability to receive intensive chemotherapy. Hence, the purpose of this study was to investigate the effects of AHCC on AML cells both in vitro and in vivo. Results showed that AHCC induced Caspase-3-dependent apoptosis in AML cell lines as well as in primary AML leukopheresis samples. Additionally, AHCC induced Caspase-8 cleavage as well as Fas and TRAIL upregulation, suggesting involvement of the extrinsic apoptotic pathway. In contrast, monocytes from healthy donors showed suppressed Caspase-3 cleavage and lower cell death. When tested in a murine engraftment model of AML, AHCC led to significantly increased survival time and decreased blast counts. These results uncover a mechanism by which AHCC leads to AML-cell specific death, and also lend support for the further investigation of AHCC as a potential adjuvant for the treatment of AML. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid lineage blasts. Due to its heterogeneity and to the high rate of acquired drug resistance and relapse, new treatment strategies are needed. Here, we demonstrate that IFNγ promotes AML blasts to act as effector cells within the context of antibody therapy. Treatment with IFNγ drove AML blasts toward a more differentiated state, wherein they showed increased expression of the M1-related markers HLA-DR and CD86, as well as of FcγRI, which mediates effector responses to therapeutic antibodies. Importantly, IFNγ was able to up-regulate CD38, the target of the therapeutic antibody daratumumab. Because the antigen (CD38) and effector receptor (FcγRI) were both simultaneously up-regulated on the AML blasts, we tested whether IFNγ treatment of the AML cell lines THP-1 and MV4-11 could stimulate them to target one another after the addition of daratumumab. Results showed that IFNγ significantly increased daratumumab-mediated cytotoxicity, as measured both by 51Cr release and lactate dehydrogenase release assays. We also found that the combination of IFNγ and activation of FcγR led to the release of granzyme B by AML cells. Finally, using a murine NSG model of subcutaneous AML, we found that treatment with IFNγ plus daratumumab significantly attenuated tumor growth. Taken together, these studies show a novel mechanism of daratumumab-mediated killing and a possible new therapeutic strategy for AML. [ABSTRACT FROM AUTHOR]

Prime-boost vaccination with recombinant (r) vaccinia(V)-CEA(6D)-TRICOM (triad of co-stimulatory molecules B7.1, ICAM-1 and LFA-3) and rFowlpox(F)-CEA(6D)-TRICOM infect antigen-presenting cells and direct expression of co-stimulatory molecules. We hypothesized that co-administration of vaccine with GM-CSF and interferon alpha (IFN-α) would have efficacy in CEA-expressing cancers. Patients with CEA-expressing cancers received the rV-CEA(6D)-TRICOM vaccine subcutaneously (s.c.) on day 1 followed by GM-CSF s.c. to the injection site on days 1-4. In Cycle 1, patients received thrice weekly s.c. injections of IFN-α-2b the week after rV-CEA(6D)-TRICOM. In Cycles 2-4, patients received thrice weekly s.c. injections of IFN-α-2b the same week that rF-CEA(6D)-TRICOM was given. The first cohort received no IFN followed by dose escalation of IFN-α in subsequent cohorts. Thirty-three patients were accrued (mean 59.8 years). Grade 3 toxicities included fatigue and hyperglycemia. Grade 4-5 adverse events (unrelated to treatment) were confusion (1), elevated aspartate transaminase (AST)/alanine transaminase (ALT) (1), and sudden death (1). No patients had a partial response, and eight patients exhibited stable disease of ≥3 months. Median progression-free survival and overall survival (OS) were 1.8 and 6.3 months, respectively. Significantly higher serum CD27 levels were observed after vaccine therapy ( p = 0.006 post 1-2 cycles, p = 0.003 post 3 cycles, p = 0.03 post 4-7 cycles) and 42 % of patients assayed developed CEA-specific T cell responses. Pre-treatment levels of myeloid-derived suppressor cells correlated with overall survival ( p = 0.04). Administration of IFN-α led to significantly increased OS ( p = 0.02) compared to vaccine alone. While the vaccine regimen produced no clinical responses, IFN-α administration was associated with improved survival. [ABSTRACT FROM AUTHOR]

Nurse-like cells (NLCs) play a central role in chronic lymphocytic leukemia (CLL) because they promote the survival and proliferation of CLL cells. NLCs are derived from the monocyte lineage and are driven toward their phenotype via contact-dependent and -independent signals from CLL cells. Because of the central role of NLCs in promoting disease, new strategies to eliminate or reprogram them are needed. Successful reprogramming may be of extra benefit because NLCs express Fc receptors (FcγRs) and thus could act as effector cells within the context of antibody therapy. IFNγ is known to promote the polarization of macrophages toward an M1-like state that is no longer tumor-supportive. In an effort to reprogram the phenotype of NLCs, we found that IFNγ up-regulated the M1-related markers CD86 and HLA-DR as well as FcγRIa. This corresponded to enhanced FcγR-mediated cytokine production as well as rituximab-mediated phagocytosis of CLL cells. In addition, IFNγ down-regulated the expression of CD31, resulting in withdrawal of the survival advantage on CLL cells. These results suggest that IFNγ can re-educate NLCs and shift them toward an effector-like state and that therapies promoting local IFNγ production may be effective adjuvants for antibody therapy in CLL. [ABSTRACT FROM AUTHOR]

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immature myeloid cells that expand in tumor-bearing hosts in response to soluble factors produced by tumor and stromal cells.MDSCexpansion has been linked to loss of immune effector cell function and reduced efficacy of immune-based cancer therapies, highlighting the MDSC population as an attractive therapeutic target. Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and IL2-inducible T-cell kinase (ITK), is in clinical use for the treatment of B-cell malignancies. Here, we report that BTK is expressed by murine and human MDSCs, and that ibrutinib is able to inhibit BTK phosphorylation in these cells. Treatment of MDSCs with ibrutinib significantly impaired nitric oxide production and cell migration. In addition, ibrutinib inhibited in vitro generation of human MDSCs and reduced mRNA expression of indolamine 2,3-dioxygenase, an immunosuppressive factor. Treatment of mice bearing EMT6 mammary tumors with ibrutinib resulted in reduced frequency of MDSCs in both the spleen and tumor. Ibrutinib treatment also resulted in a significant reduction of MDSCs in wild-type mice bearing B16F10 melanoma tumors, but not in X-linked immunodeficiency mice (XID) harboring a BTK mutation, suggesting that BTK inhibition plays an important role in the observed reduction of MDSCs in vivo. Finally, ibrutinib significantly enhanced the efficacy of anti-PD-L1 (CD274) therapy in a murine breast cancer model. Together, these results demonstrate that ibrutinib modulates MDSC function and generation, revealing a potential strategy for enhancing immune-based therapies in solid malignancies. [ABSTRACT FROM AUTHOR]

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγRmediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis( acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function. [ABSTRACT FROM AUTHOR]

Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STATI and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR. and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 nig sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-a, and evaluated for phosphorylation of STATI and STAT5. Pretreatment of PBMCs with 10 µM sorafenib decreased STATI and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 µ,M), IL-2 (2-24 nM), and IFN-a (10¹-106 U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MlPl-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells. [ABSTRACT FROM AUTHOR]

FcγRs are critical mediators of mAb cancer therapies, because they drive cytotoxic processes upon binding of effector cells to opsonized targets. Along with NK cells, monocytes are also known to destroy Ab-coated targets via Ab-dependent cellular cytotoxicity (ADCC). However, the precise mechanisms by which monocytes carry out this function have remained elusive. In this article, we show that human monocytes produce the protease granzyme B upon both FcγR and TLR8 activation. Treatment with TLR8 agonists elicited granzyme B and also enhanced FcγR-mediated granzyme B production in an additive fashion. Furthermore, monocyte-mediated ADCC against cetuximab-coated tumor targets was enhanced by TLR8 agonist treatment, and this enhancement of ADCC required granzyme B. Hence we have identified granzyme B as an important mediator of FcγR function in human monocytes and have uncovered another mechanism by which TLR8 agonists may enhance FcγR-based therapies. [ABSTRACT FROM AUTHOR]

Glioblastoma is a devastating disease, and there is an urgent need to develop novel therapies, such as oncolytic HSV1 (OV) to effectively target tumor cells. OV therapy depends on tumor-specific replication leading to destruction of neoplastic tissues. Host responses that curtail virus replication limit its efficacy in vivo. We have previously shown that cysteine-rich 61 protein (CCN1) activates a type 1 IFN antiviral defense response in glioblastoma cells. Incorporating TCGA data, we found CCN1 expression to be a negative prognostic factor for glioblastoma patients. Based on this, we used neutralizing antibodies against CCN1 to investigate its effect on OV therapy. Use of an anti-CCN1 antibody in mice bearing glioblastomas treated with OV led to enhanced virus expression along with reduced immune cell infiltration. OV-induced CCN1 increases macrophage migration toward infected glioblastoma cells by directly binding macrophages and also by enhancing the proinflammatory activation of macrophages inducing MCP-1 expression in glioblastoma cells. Activation of macrophages by CCN1 also increases viral clearance. Neutralization of integrin αMβ2 reversed CCN1-induced macrophage activation and migration, and reduced MCP-1 expression by glioblastoma cells. Our findings reveal that CCN1 plays a novel role in pathogen clearance; increasing macrophage infiltration and activation resulting in increased virus clearance in tumors. [ABSTRACT FROM AUTHOR]

Sepsis rapidly activates the host inflammatory response and acute phase response. Severe sepsis, complicated by multiple organ failure, is associated with overwhelming inflammation and high mortality. We previously observed that zinc (Zn) deficiency significantly increases mortality in a mouse model of polymicrobial sepsis due to over-activation of the inflammatory response. In order to identify potential mechanisms that account for Zn-responsive effects, we generated whole exome expression profiles from the lung tissue of septic mice that were maintained on Zn modified diets. Based on systems analysis, we observed that Zn deficiency enhances the acute phase response and particularly the JAK-STAT3 pathway, resulting in increased serum amyloid A production. In vitro studies of primary hepatocytes and HepG2 cells substantiated that Zn-deficiency augments serum amyloid A production through up-regulation of the JAK-STAT3 and NF-κB pathways. In contrast, Zn inhibited STAT3 activation through the up-regulation of SHP1 activity. Collectively, these findings demonstrate that Zn deficiency enhances the acute phase response through up-regulation of the JAK-STAT3 pathway, thereby perpetuating increased inflammation that may lead to increased morbidity and mortality in response to sepsis. [ABSTRACT FROM AUTHOR]

Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt- 1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing themechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-αB, we found that a microRNA, miR181a, negatively regulates FcR- mediated sFlt-1 production and that NF-αB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy. [ABSTRACT FROM AUTHOR]

Fcγ receptor (FcγR) clustering on monocytes/macrophages results in phagocytosis and inflammatory cytokine production, which serve to eliminate antibody-opsonized targets and activate neighboring immune cells. Toll-like receptor 2 (TLR2), which recognizes a range of both bacterial and fungal components, elicits strong proinflammatory responses in these cells when stimulated by ligands, either natural or synthetic. Thus, we explored the possibility that TLR2 agonists could strengthen FcγR activity within the context of antibody therapy. Human peripheral blood monocytes treated with the TLR2 agonist Pam2CSK4 showed significantly enhanced FcγR-mediated cytokine production as well as phagocytic ability. An examination of the molecular mechanism behind this enhancement revealed increased expression of both FcγRIIa and the common γ subunit following Pam2CSK4 treatment. Interestingly however, expression of the inhibitory receptor FcγRIIb was also modestly increased. Further investigation revealed that Pam2CSK4 also dramatically decreased the expression of SHIP, the major mediator of FcγRIIb inhibitory activity. Using a murine Her2/neu solid tumor model of antibody therapy, we found that Pam2CSK4 significantly enhanced the ability of anti-Her2 antibody to reduce the rate of tumor growth. To verify that the FcγR enhancement was not unique to the diacylated Pam2CSK4, we also tested Pam3CSK4, a related triacylated TLR2 agonist. Results showed significant enhancement in FcγR function and expression. Taken together, these findings indicate that TLR2 activation can positively modulate FcγR and suggest that TLR2 agonists should be considered for testing as adjuvants for antitumor antibody therapy. [ABSTRACT FROM AUTHOR]

CD20 is a widely validated, B cell-specific target for therapy in B cell malignancies. Rituximab is an anti-CD20 Ab that prolongs survival of chronic lymphocytic leukemia (CLL) patients when combined with chemotherapy. Ofatumumab and GA101 (obinutuzumab) are CD20-directed Abs currently being developed as alternative agents to rituximab in CLL based upon different properties of enhanced direct cell death, NK cell-mediated Ab-dependent cellular cytotoxicity, or complement-dependent cytotoxicity. Despite widespread study, ofatumumab and GA101 have not been compared with each other, nor studied for their interactions with monocytes and macrophages which are critical for the efficacy of anti-CD20 Abs in murine models. In CLL cells, we show that direct cell death and complement-dependent cytotoxicity are greatest with GA101 and ofatumumab, respectively. GA101 promotes enhanced NK cell activation and Ab-dependent cellular cytotoxicity at high Ab concentrations. Ofatumumab elicits superior Ab-dependent cellular phagocytosis with monocyte-derived macrophages. GA101 demonstrated reduced activation of monocytes with diminished pERK, TNF-α release, and FcγRIIa recruitment to lipid rafts. These data demonstrate that GA101 and ofatumumab are both superior to rituximab against CLL cells via different mechanisms of potential tumor elimination. These findings bear relevance to potential combination strategies with each of these anti-CD20 Abs in the treatment of CLL. [ABSTRACT FROM AUTHOR]

Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation. [ABSTRACT FROM AUTHOR]

Burkholderia cenocepacia, the causative agent of cepacia syndrome, primarily affects cystic fibrosis patients, often leading to death. In the lung, epithelial cells serve as the initial barrier to airway infections, yet their responses to B. cenocepacia have not been fully investigated. Here, we examined the molecular responses of human airway epithelial cells to B. cenocepacia infection. Infection led to early signaling events such as activation of Erk, Akt, and NF-κB. Further, TNFα, IL-6, IL-8, and IL-1β were all significantly induced upon infection, but no IL-1β was detected in the supernatants. Because caspase-1 is required for IL-1β processing and release, we examined its expression in airway epithelial cells. Interestingly, little to no caspase-1 was detectable in airway epithelial cells. Transfection of caspase-1 into airway epithelial cells restored their ability to secrete IL-1β following B. cenocepacia infection, suggesting that a deficiency in caspase-1 is responsible, at least in part, for the attenuated IL-1β secretion. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRs) are small, noncodlng RNA molecules with important regulatory functions whose role in regulating natural killer (NK) cell biology is not well defined. Here, we show that miR-155 is synergistically induced in primary human NK cells after costimulation with IL-12 and IL-18, or with IL-12 and CD16 clustering. Over-expression of miR-155 enhanced induction of IFN-7 by IL-12 and IL-18 or CD16 stimulation, whereas knockdown of miR155 or its disruption suppressed IFN-7 induction in monokine and/or CD16-stimulated NK cells. These effects on the regulation of NK cell IFN-7 expression were found to be mediated at least in part via miR-155's direct effects on the inositol phosphatase SHIP1. Consistent with this, we observed that modulation of miR-155 overrides IL-12 and IL-18-mediated regulation of SHIP1 expression in NK cells. Collectively, our data indicate that miR-155 expression is regulated by stimuli that strongly induce IFN-7 in NK cells such as IL-12, IL-18, and CD16 activation, and that miR-155 functions as a positive regulator of IFN-7 production in human NK cells, at least in part via down-regulating SHIP1. These findings may have clinical relevance for targeting miR-155 in neoplastic disease. [ABSTRACT FROM AUTHOR]

Airway epithelial cells in the lung are the first line of defense against pathogens and environmental pollutants. Inhalation of the environmental pollutant cadmium has been linked to the development of lung cancer and chronic obstructive pulmonary disease, which are diseases characterized by chronic inflammation. To address the role of airway epithelial cells in cadmium-induced lung inflammation, we investigated how cadmium regulates secretion of interleukin 8 (IL-8) by airway epithelial cells. We show that exposure of human airway epithelial cells to subtoxic doses of cadmium in vitro promotes a characteristic inflammatory cytokine response consisting of IL-8, but not IL-1β or tumor necrosis factor-alpha. We also found that intranasal delivery of cadmium increases lung levels of the murine IL-8 homologs macrophage inflammatory protein-2 and keracinocyte-dervied chemokine and results in an influx of Gr1+ cells into the lung. We determined that inhibition of the nuclear factor-κB (NF-κB) pathway had no effect on cadmium-induced IL-8 secretion by human airway epithelial cells, suggesting that IL-8 production was mediated through an NF-κB–independent pathway. Mitogen-activated protein kinases (MAPKs) are often involved in proinflammatory signaling. Cadmium could activate the main MAPKs (i.e., p38, JNK, and Erk1/2) in human airway epithelial cells. However, only pharmacological inhibition of Erk1/2 pathway or knockdown of the expression of Erk1 and Erk2 using small interfering RNAs suppressed secretion of IL-8 induced by cadmium. Our findings identify cadmium as a potent activator of the proinflammatory cytokine IL-8 in lung epithelial cells and reveal for the first time the role of an NF-κB–independent but Erk1/2-dependent pathway in cadmium-induced lung inflammation. [ABSTRACT FROM PUBLISHER]

Abstract: Cadmium is a toxic metal present in the environment and its inhalation can lead to pulmonary disease such as lung cancer and chronic obstructive pulmonary disease. These lung diseases are characterized by chronic inflammation. Here we show that exposure of human airway epithelial cells to cadmium promotes a polarized apical secretion of IL-6 and IL-8, two pivotal pro-inflammatory cytokines known to play an important role in pulmonary inflammation. We also determined that two distinct pathways controlled secretion of these proinflammatory cytokines by human airway epithelial cells as cadmium-induced IL-6 secretion occurs via an NF-κB dependent pathway, whereas IL-8 secretion involves the Erk1/2 signaling pathway. Interestingly, the natural antioxidant curcumin could prevent both cadmium-induced IL-6 and IL-8 secretion by human airway epithelial cells. In conclusion, curcumin could be used to prevent airway inflammation due to cadmium inhalation. [Copyright &y& Elsevier]

The environmental bacterium Burkholderia cenocepacia causes opportunistic lung infections in immunocompromised individuals, particularly in patients with cystic fibrosis. Infections in these patients are associated with exacerbated inflammation leading to rapid decay of lung function, and in some cases resulting in cepacia syndrome, which is characterized by a fatal acute necrotizing pneumonia and sepsis. B. cenocepacia can survive intracellularly in macrophages by altering the maturation of the phagosome, but very little is known on macrophage responses to the intracellular infection. In this study, we have examined the role of the PI3K/Akt signaling pathway in B. cenocepacia-infected monocytes and macrophages. We show that PI3K/Akt activity was required for NF-κB activity and the secretion of proinflammatory cytokines during infection with B. cenocepacia. In contrast to previous observations in epithelial cells infected with other Gram-negative bacteria, Akt did not enhance IκB kinase or NF-κB p65 phosphorylation, but rather inhibited GSK3β, a negative regulator of NF-κB transcriptional activity. This novel mechanism of modulation of NF-κB activity may provide a unique therapeutic target for controlling excessive inflammation upon B. cenocepacia infection. [ABSTRACT FROM AUTHOR]

SHIP and SHIP-2 are inositol phosphatases that regulate Fc&ggr;R-mediated phagocytosis through catalytic as well as noncatalytic mechanisms. In this study we have used two-dimensional fluorescence difference gel electrophoresis (DIGE) analysis to identify downstream signaling proteins that uniquely associate with SHIP or SHIP-2 upon Fc&ggr;R clustering in human monocytes. We identified LyGDI as a binding partner of SHIP, associating inducibly with the SHIP/Grb2/Shc complex. Immunodepletion and competition experiments with recombinant SHIP domains revealed that Grb2 and the proline-rich domain of SHIP were necessary for SHIP-LyGDI association. Functional studies in primary human monocytes showed that LyGDI sequesters Rac in the cytosol, preventing it from localizing to the membrane. Consistent with this, suppression of LyGDI expression resulted in significantly enhanced Fc&ggr;R-mediated phagocytosis. [ABSTRACT FROM AUTHOR]

IL-15 may have a role in the development of T cell large granular lymphocyte (T-LGL) or NKT leukemias. However, the mechanisms of action and the identity of the cell subset that undergoes leukemic transformation remain elusive. Here we show that in both mice and humans, NKp46 expression marks a minute population of WT NKT cells with higher activity and potency to become leukemic. Virtually 100% of T-LGL leukemias in IL-15 transgenic mice expressed NKp46, as did a majority of human T-LGL leukemias. The minute NKp46- NKT population, but not the NKp46 NKT population, was selectively expanded by overexpression of endogenous IL-15. Importantly, IL-15 transgenic NKp46 NKT cells did not become NKp46+ in vivo, suggesting that NKp46+ T-LGL leukemia cells were the malignant counterpart of the minute WT NKp4+ NKT population. Mechanistically, NKp46+ NKT cells possessed higher responsiveness to IL-is in vitro and in vivo compared with that of their NKp46- NKT counterparts. Furthermore, interruption of IL-15 signaling using a neutralizing antibody could prevent LGL leukemia in IL-15 transgenic mice. Collectively, our data demonstrate that NKp46 identifies a functionally distinct NKT subset in mice and humans that appears to be directly susceptible to leukemic transformation when IL-15 is overexpressed. Thus, IL-15 signaling and NKp46 may be useful targets in the treatment of patients with T-LGL or NKT leukemia. [ABSTRACT FROM AUTHOR]

The intracellular Gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/ macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 39UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated proinflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced proinflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP downregulation and pro-inflammatory response and may account for the virulence of Type A Francisella. [ABSTRACT FROM AUTHOR]

The article presents a study which hypothesized that the mitochondrial fraction of the cell contributes to the monocyte activation by necrotic cells. Result shows that mitochondrial cell fractions activated monocytes and mitochondria from Rho(0) cells induced minimal monocyte Interleukin-8 release. They concluded that interactions between mitochondrial N-formyl peptides and formyl peptide receptor (FPR) in the presence of other mitochondrial antigens activated monocytes by necrotic cells.

Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Nlrc4-/- (Ipaf-/-), and caspase-1-/- derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that, upon infection of murine macrophages, caspase-7 was activated downstream of the Nlrc4 inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin and required a functional Naip5. Remarkably, mice lacking caspase-7 and its macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7-/- macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Nlrc4 inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium. [ABSTRACT FROM AUTHOR]

Antibody-based therapies, such as rituximab and alemtuzumab, have contributed significantly to the treatment of Chronic Lymphocytic leukaemia (CLL). The CD40 antigen is expressed predominantly on B-cells and represents a potential target for immune-based therapies. SGN-40 is a humanized IgG1 monoclonal antibody currently in Phase I/II clinical trials for indolent lymphomas, diffuse large B cell lymphomas and Multiple Myeloma. Its biological effect on CLL cells has not been studied. The present study demonstrated that SGN-40 mediated modest apoptosis in a subset of patients with secondary cross-linking but did not mediate complement-dependent cytotoxicity. SGN-40 also mediated antibody-dependent cellular cytotoxicity (ADCC) predominantly through natural killer (NK) cells. Previous studies by our group and others have demonstrated that lenalidomide upregulates CD40 expression on primary B CLL cells and activates NK-cells. We therefore examined for the combinatorial effect of lenalidomide and SGN-40 and demonstrated that both enhanced direct apoptosis and ADCC against primary CLL B cells. These data together provide justification for clinical trials of SGN-40 and lenalidomide in combination for CLL therapy. [ABSTRACT FROM AUTHOR]

Macrophages are important effectors in the clearance of antibody-coated tumor cells. However, the signaling pathways that regulate macrophage-induced ADCC are poorly defined. To understand the regulation of macrophage-mediated ADCC, we used human B cell lymphoma coated with Rituximab as the tumor target and murine macrophages primed with IFNγ as the effectors. Our data demonstrate that the PtdIns 3-kinase/Akt pathway is activated during macrophage-induced ADCC and that the inhibition of PtdIns 3-kinase results in the inhibition of macrophage-mediated cytotoxicity. Interestingly, downstream of PtdIns 3-kinase, expression of constitutively active Akt (Myr-Akt) in macrophages significantly enhanced their ability to mediate ADCC. Further analysis revealed that in this model, macrophage-mediated ADCC is dependent upon the release of nitric oxide (NO). However, the PtdIns 3-kinase/Akt pathway does not appear to regulate NO production. An examination of the role of the PtdIns 3-kinase/Akt pathway in regulating conjugate formation indicated that macrophages treated with an inhibitor of PtdIns 3-kinase fail to polarize the cytoskeleton at the synapse and show a significant reduction in the number of conjugates formed with tumor targets. Further, inhibition of PtdIns 3-kinase also reduced macrophage spreading on Rituximab-coated surfaces. On the other hand, Myr-Akt expressing macrophages displayed a significantly greater ability to form conjugates with tumor cells. Taken together, these findings illustrate that the PtdIns 3-kinase/Akt pathway plays a critical role in macrophage ADCC through its influence on conjugate formation between macrophages and antibody-coated tumor cells. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a gram-negative facultative bacterium that causes the disease tularemia, even upon exposure to low numbers of bacteria. One critical characteristic of Francisella is its ability to dampen or subvert the host immune response. In order to help understand the mechanisms by which this occurs, we performed Affymetrix microarray analysis on transcripts from blood monocytes infected with the virulent Type A Schu S4 strain. Results showed that expression of several host response genes were reduced such as those associated with interferon signaling, Toll-like receptor signaling, autophagy and phagocytosis. When compared to microarrays from monocytes infected with the less virulent F. tularensis subsp. novicida, we found qualitative differences and also a general pattern of quantitatively reduced pro-inflammatory signaling pathway genes in the Schu S4 strain. Notably, the PI3K / Akt1 pathway appeared specifically down-regulated following Schu S4 infection and a concomitantly lower cytokine response was observed. This study identifies several new factors potentially important in host cell subversion by the virulent Type A F. tularensis that may serve as novel targets for drug discovery. [ABSTRACT FROM AUTHOR]

Abstract: Tularemia is a zoonotic disease caused by the Gram-negative intracellular pathogen Francisella tularensis. These bacteria evade phagolysosomal fusion, escape from the phagosome and replicate in the host cell cytoplasm. IFNγ has been shown to suppress the intra-macrophage growth of Francisella through both nitric oxide-dependent and -independent pathways. Since Francisella is known to subvert host immune responses, we hypothesized that this pathogen could interfere with IFNγ signaling. Here, we report that infection with Francisella suppresses IFNγ-induced STAT1 expression and phosphorylation in both human and murine mononuclear phagocytes. This suppressive effect of Francisella is independent of phagosomal escape or replication and is mediated by a heat-stable and constitutively expressed bacterial factor. An analysis of the molecular mechanism of STAT1 inhibition indicated that expression of SOCS3, an established negative regulator of IFNγ signaling, is highly up-regulated during infection and suppresses STAT1 phosphorylation. Functional analyses revealed that this interference with IFNγ signaling is accompanied by the suppression of IP-10 production and iNOS induction resulting in increased intracellular bacterial survival. Importantly, the suppressive effect on IFNγ-mediated host cell protection is most effective when IFNγ is added post infection, suggesting that the bacteria establish a permissive environment within the host cell. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a highly infectious, Gram-negative intra-cellular pathogen that can cause the zoonotic disease tularemia. Although the receptors critical for internalization of Francisella by macrophages are beginning to be defined, the identity of the downstream signaling pathways essential for the engulfment are not yet identified. In this study we have tested the role of Syk in the phagocytosis of Francisella. We report that Syk is activated during Francisella infection and is critical for the uptake of the organisms. Pharmacologic inhibition of Syk almost completely abrogated uptake, whereas the overexpression of Syk significantly enhanced uptake. However, Syk appears to be dispensable during initial host–pathogen contact. Further analyses of the molecular mechanism of Syk influence on Francisella uptake revealed that the MAPK Erk but not the phosphatidylinositol 3 kinase (PI3K)/Akt pathway is the downstream effector of Syk. Thus, the inhibition of Erk in Syk-overexpressing cells or the inhibition of Syk in Erk-overexpressing cells led to a significant attenuation of uptake. Collectively, these data identify Syk and Erk as key players in the phagocytosis of Francisella. [Copyright &y& Elsevier]

Abstract: We previously demonstrated that monocytes produce IL-23 during Francisella infection, and that IL-23 induces IFNγ from NK cells. Here, we demonstrate that IFNγ-priming of monocytes enhances IL-23 production during Francisella infection. This effect was seen on the IL12/23 p40 subunit. Induction of IL-12/23 p40 is reported to be enhanced by IRF-1 and IRF-8. Consistently, microarray analysis of IFNγ-treated monocytes revealed a significant induction of the IRFs. Interestingly, IFNγ-primed monocytes produced IL-12 p70, a more potent inducer of IFNγ than IL-23. We propose that there exists an amplification loop between monocyte IL-23 and NK/T cell IFNγ that leads to IL-12 p70 production. [Copyright &y& Elsevier]

A combinatorial phosphotyrosyl (pY) peptide library was screened to determine the amino acid preferences at the pY+4 to pY + 6 positions for the four SH2 domains of protein-tyrosine phosphatases SHP-1 and SHP-2. Individual binding sequences selected from the library were resynthesized and their binding affinities and specificities to various SH2 domains were further evaluated by SPR studies, stimulation of SHP-1 and SHP-2 phosphatase activity, and in vitro pulldown assays. These studies reveal that binding of a pY peptide to the N-SH2 domain of SHP-2 is greatly enhanced by a large hydrophobic residue (Trp, Tyr, Met, or Phe) at the pY+4 and/or pY+5 positions, whereas binding to SHP-1 N-SH2 domain is enhanced by either hydrophobic or positively charged residues (Arg, Lys, or His) at these positions. Similar residues at the pY+4 to pY+6 positions are also preferred by SHP-1 and SHP-2 C-SH2 domains, although their influence on the overall binding affinities is much smaller compared with the N-SH2 domains. A structural model was generated to qualitatively interpret the contribution of the pY+4 and pY+5 residues to the overall binding affinity. Examination of pY motifs from known SHP-1 and SHP-2-binding proteins shows that many of the pY motifs contain a hydrophobic or positively charged residue(s) at the pY+4 and pY+5 positions. [ABSTRACT FROM AUTHOR]

Francisella tularensis, a Gram-negative facultative intracellular pathogen infecting principally macrophages and monocytes, is the etiological agent of tularemia. Macrophage responses to F. tularensis infection include the production of pro-inflammatory cytokines such as interleukin (IL)-12, which is critical for immunity against infection. Molecular mechanisms regulating production of these inflammatory mediators are poorly understood. Herein we report that the SH2 domain-containing inositol phosphatase (SHIP) is phosphorylated upon infection of primary murine macrophages with the genetically related F. novicida, and negatively regulates F. novicida-induced cytokine production. Analyses of the molecular details revealed that in addition to activating the MAP kinases, F. novicida infection also activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in these cells. Interestingly, SHIP-deficient macrophages displayed enhanced Akt activation upon F. novicida infection, suggesting elevated PI3K-dependent activation pathways in absence of SHIP. Inhibition of PI3K/Akt resulted in suppression of F. novicida-induced cytokine production through the inhibition of NFκB. Consistently, macrophages lacking SHIP displayed enhanced NFκB-driven gene transcription, whereas overexpression of SHIP led to decreased NFκB activation. Thus, we propose that SHIP negatively regulates F. novicida-induced inflammatory cytokine response by antagonizing the PI3K/Akt pathway and suppressing NFκB-mediated gene transcription. A detailed analysis of phosphoinositide signaling may provide valuable clues for better understanding the pathogenesis of tularemia. [ABSTRACT FROM AUTHOR]

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 mm of global ischemia at 37°C followed by 45 mm of reperfusion. Hearts were perfused with PC (10 μ.M) or Spirulina preparation (SP, 50 mg/I) for 15 mm before the onset of ischemia and throughout reperfusion. After 45 mm of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK 1⁄2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1⁄2. [ABSTRACT FROM AUTHOR]

Abstract: The bacterial endotoxin lipopolysaccharide (LPS), is a potent inducer of the inflammatory response. Previous studies demonstrated that LPS-induced toxicity is reversed upon FcγR clustering by IgG immune complexes (IC) through upregulation of the anti-inflammatory cytokine IL-10. The PI3K-Akt pathway is also reported to reverse LPS-induced inflammation. In this study, we have examined the role of Akt in LPS-induced IL-10 production. First, we compared Akt activation in macrophages stimulated with either LPS alone, or with a combination of LPS and ICs. Our experiments revealed that while Akt was activated under both conditions, the level of activation was significantly higher in cells stimulated with LPS and ICs, suggesting that Akt may be involved in IC-induced upregulation of IL-10 production. Using several independent models we have then tested the notion that enhanced Akt activation may lead to enhanced LPS-induced IL-10 production. Over-expression of constitutively active Myr-Akt in the mouse macrophage cell line Raw 264.7 led to significant increase in IL-10 production in response to LPS. In addition, down-regulation of Akt by siRNA resulted in a decrease in LPS-induced IL-10 production. Peritoneal macrophages from transgenic mice with macrophage-specific expression of Myr-Akt produced significantly higher levels of IL-10 when stimulated with LPS, compared to their wild-type counterparts. Consistent with this observation, serum levels of IL-10, post-LPS challenge, was higher in the Myr-Akt transgenic mice compared to the wild-type mice. Taken together, these data demonstrate that Akt plays a critical role in LPS-induced production of IL-10. [Copyright &y& Elsevier]

Abstract: The inhibitory receptor FcγRIIb becomes tyrosine phosphorylated and associates with the inositol phosphatase SHIP to downregulate phagocytosis. The two splice variants of FcγRIIb, b1 and b2, are differentially expressed in hematopoetic cells. Both isoforms of FcγRIIb are expressed in human myeloid cells although FcγRIIb2 predominates. In murine B cells FcγRIIb2 associates with clathrin-coated pits and undergoes endocytosis, whereas FcγRIIbl is excluded from the coated pits, indicating that the two isoforms serve partially differing functions. In humans, there are conflicting reports with regard to the ability of FcγRIIb2 to become tyrosine phosphorylated, and the functional capacities of the two isoforms are poorly understood. We, and others, have previously reported that the expression of FcγRIIb is upregulated in human monocytes by the anti-inflammatory cytokine IL-4. Here, we extend these findings to demonstrate that the IL-4-induced upregulation of FcγRIIb is synergistically enhanced by the addition of IL-10, both at the protein and the mRNA level. The upregulated receptors are functional as assessed by their ability to become tyrosine phosphorylated and to downregulate phagocytosis. Interestingly, both b1 and b2 isoforms are upregulated by anti-inflammatory cytokines. Transfection experiments expressing human FcγRIIbl or b2 in Raw 264.7 murine macrophage cells revealed that both isoforms are tyrosine phosphorylated and promote SHIP phosphorylation. Finally, both b1 and b2 isoforms of FcγRIIb downregulate phagocytosis to a similar extent. Thus we conclude that FcγRIIbl and b2 are both functional inhibitory receptors in the phagocytic process. [Copyright &y& Elsevier]

A general, combinatorial library method for the rapid identification of high-affinity peptide ligands of protein modular domains is reported. The validity of this method has been demonstrated by determining the sequence specificity of four Src homology 2 (SH2) domains derived from protein tyrosine phosphatase SHP- 1 and SHP-2 and inositol phosphatase SHIP. A phosphotyrosyl (pY) peptide library was screened against the SH2 domains, and the beads that carry high-affinity ligands of the SH2 domains were identified and peptides were sequenced by partial Edman degradation and mass spectrometry. The results reveal that the N-terminal SH2 domain of SHP-2 is capable of recognizing four different classes of pY peptides. Binding competition studies suggest that the four classes of pY peptides all bind to the same site on the SH2 domain surface. The C-terminal SH2 domains of SHP-1 and SHP-2 and the SHIP SH2 domain each bind to pY peptides of a single consensus sequence. Database searches using the consensus sequences identified most of the known as well as many potential interacting proteins of SHP-1 and/or SHP-2. Several proteins are found to bind to the SH2 domains of SHP-1 and SHP-2 through a new, nonclassical ITIM motif, (V/I/L)XpY(M/L/F)XP, which corresponds to the class IV peptides selected from the pY library. The combinatorial library method should be generally applicable to other protein domains. [ABSTRACT FROM AUTHOR]

Fcγ receptor (FcγR) clustering by immune complexes activates multiple signaling pathways leading to phagocytosis. We and others have previously reported that Akt is phosphorylated in response to FcγR clustering. However, the functional consequence of Akt activation by FcγR is not known. Using Raw 264.7 macrophage cells transfected to overexpress either constitutively active myristoylated (Myr)-Akt or a dominant-negative CAAX-Akt and bone marrow macrophages (BMMs) from wild-type and transgenic mice expressing macrophage-specific Myr-Akt, we analyzed the function of Akt in phagocytosis. We report that overexpression of Myr-Akt resulted in significant increase in phagocytic efficiency, whereas CAAX-Akt down-regulated phagocytosis in Raw 264.7 cells. Likewise BMMs expressing Myr-Akt dis- played enhanced phagocytic ability. Analyzing the downstream effectors of Akt, we demonstrate that p70S6 kinase is constitutively phosphorylated in Myr-Akt-expressing BMMs. p70S6 kinase is reported to influence actin cytoskeleton and cell migration, suggesting that Akt may influence phagocytosis through the activation of p70S6 kinase. Consistent with this, overexpression of either wild-type or constitutively active but not a kinase-inactive p70S6 kinase in Raw 264.7 cells significantly enhanced phagocytosis. Likewise suppression of p70S6 kinase with rapamycin down-regulated phago- cytic efficiency conferred by the expression of constitutively active Akt. These findings demonstrate a novel role for Akt in phagocytosis through the activation of p70S6 kinase. [ABSTRACT FROM AUTHOR]

FcγRIIa is a low affinity IgG receptor uniquely expressed in human cells that promotes phagocytosis of immune complexes and induces inflammatory cytokine gene transcription. Recent studies have revealed that phagocytosis initiated by FcγRIIa is tightly controlled by the inositol phosphatase SHIP-1, and the proteintyrosine phosphatase SHP-1. Whereas the molecular nature of SHIP-1 involvement with FcγRIIa has been well studied, it is not clear how SHP-1 is activated by FcγRIIa to mediate its regulatory effect. Here we report that FcγRIIa clustering induces SHP-1 phosphatase activity in THP-1 cells. Using synthetic phosphopeptides, and stable transfectants expressing immunoreceptor tyrosine-based activation motif (ITAM) tyrosine mutants of FcγRIIa, we demonstrate that SHP-1 associates with the phosphorylated amino-terminal ITAM tyrosine of FcγRIIa, whereas the tyrosine kinase Syk associates with the carboxyl-terminal ITAM tyrosine. Association of SHP-1 with FcγRIIa ITAM appears to suppress total cellular tyrosine phosphorylation. Furthermore, FcγRIIa clustering results in the association of SHP-1 with key signaling molecules such as Syk, p85 subunit of PtdIns 3-kinase, and p62dok, suggesting that these molecules may be substrates of SHP-1 in this system. Finally, overexpression of wild-type SHP-1 but not catalytically deficient SHP-1 led to a down-regulation of NFκB-dependent gene transcription in THP-1 cells activated by clustering FcγRIIa. [ABSTRACT FROM AUTHOR]

Analyzes the expression patterns, molecular mechanism of activation, and function of the inositol 5'-phosphatase SHIP-2 in human myeloid cell FCγ receptor (FcγR) signaling. Association of SHIP-2 with the phosphorylated immunoreceptor tyrosine-based activation motif of FcγRIIa; Induction of SHIP-2 phosphorylation by FcγRI clustering in THP-1 cells.

Background: Tumor-associated macrophages (TAM) are expanded and exhibit tumor-promoting properties within the tumor microenvironment. Current methods to study TAM have not been replicated across cancer types and often do not include exogenous growth factors from the tumor, a key factor in TAM differentiation in vivo. Methods: In this study, an in vitro method to generate monocyte- derived TAM using tumor- conditioned media (TCM) and a cytokine cocktail containing IL-4, IL-10, and M-CSF was utilized to study the phenotype, morphology, and function of TAM across multiple cancer types. TCM was generated from two breast cancer cell lines and an Epstein-Barr virus-positive lymphoma cell line. The properties of in vitro generated TAM were compared to in vitro generated M1 and M2- like macrophages and TAM isolated from patients with cancer. Results: TAM generated in this fashion displayed an increase in CD163/CD206 co-expression compared to M2- like macrophages (87 and 36%, respectively). TAM generated in vitro exhibited increased transcript levels of the functional markers IL-6, IL-10, CCL2, c-Myc, iNOS, and arginase compared to in vitro generated M2-like macrophages. Functionally, in vitro generated TAM inhibited the proliferation of T cells (47% decrease from M1-like macrophages) and the production of IFN-γ by natural killer cells was inhibited (44%) when co-cultured with TAM. Furthermore, in vitro generated TAM secreted soluble factors that promote the growth and survival of tumor cells. Conclusions: Limited access to patient TAM highlights the need for methods to generate TAM in vitro. Our data confirm that monocyte-derived TAM can be generated reliably using TCM plus the cytokine cocktail of IL-4, IL-10, and M-CSF. Given the ability of TAM to inhibit immune cell function, continued study of methods to deplete or deactivate TAM in the setting of cancer are warranted. [ABSTRACT FROM AUTHOR]

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of early myeloid cells that accumulate in the blood and tumors of patients with cancer. MDSC play a critical role during tumor evasion and promote immune suppression through variety of mechanisms, such as the generation of reactive oxygen and nitrogen species (ROS and RNS) and cytokines. AMPactivated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that regulates energy homeostasis and metabolic stress. However, the role of AMPK in the regulation of MDSC function remains largely unexplored. This study was designed to investigate whether treatment of MDSC with OSU-53, a PPAR-inactive derivative that stimulates AMPK kinase, can modulate MDSC function. Our results demonstrate that OSU-53 treatment increases the phosphorylation of AMPK, significantly reduces nitric oxide production, inhibits MDSC migration, and reduces the levels of IL-6 in murine MDSC cell line (MSC2 cells). OSU53 treatment mitigated the immune suppressive functions of murine MDSC, promoting T-cell proliferation. Although OSU-53 had a modest effect on tumor growth in mice inoculated with EMT-6 cells, importantly, administration of OSU53 significantly (p< 0.05) reduced the levels of MDSC in the spleens and tumors. Furthermore, mouse MDSC from EMT-6 tumor-bearing mice and human MDSC isolated from melanoma patients treated with OSU-53 showed a significant reduction in the expression of immune suppressive genes iNOS and arginase. In summary, these results demonstrate a novel role of AMPK in the regulation of MDSC functions and provide a rationale of combining OSU-53 with immune checkpoint inhibitors to augment their response in cancer patients. [ABSTRACT FROM PUBLISHER]

Objective: To examine the signaling pathways critical for macrophage cytotoxicity to antibody-coated tumor targets. Methods: Using a human B cell lymphoma cell line coated with Rituximab as a tumor model the cytotoxic ability of murine macrophages was measured by 51Cr-release assays. In addition, biochemical, pharmacological and genetic approaches were used to identify molecular mechanisms of macrophage cytotoxicity. Findings: We first demonstrate that interaction of macrophages with antibody-coated tumor targets leads to the activation of multiple signaling events including the activation of tyrosine kinases and PI3K. Inhibition of PI3K activation completely abolished cytotoxicity indicating that the PI3K/Akt pathway is necessary for macrophage ADCC. Consistent with this, murine peritoneal macrophages expressing overactive Akt showed significantly enhanced ADCC. Further, studies showed that in this model of macrophage ADCC, cytotoxicity is dependent upon release of nitric oxide (NO). We hypothesize that Akt enhances macrophage cytotoxicity by promoting. NO production. We are currently testing this hypothesis. Conclusion: Together these findings illustrate a novel role for Akt in the clearance of antibody-coated tumor cells, and suggest that manipulation of Akt may enhance the efficacy of monoclonal antibody therapy for cancer. [ABSTRACT FROM AUTHOR]