Cocaine Enhances HIV-1 Infectivity in Monocyte Derived Dendritic Cells by Suppressing microRNA-155

Cocaine and other drugs of abuse increase HIV-induced immunopathogenesis; and neurobiological mechanisms of cocaine addiction implicate a key role for microRNAs (miRNAs), single-stranded non-coding RNAs that regulate gene expression and defend against viruses. In fact, HIV defends against miRNAs by actively suppressing the expression of polycistronic miRNA cluster miRNA-17/92, which encodes miRNAs including miR-20a. IFN-g production by natural killer cells is regulated by miR-155 and this miRNA is also critical to dendritic cell (DC) maturation. However, the impact of cocaine on miR-155 expression and subsequent HIV replication is unknown. We examined the impact of cocaine on two miRNAs, miR-20a and miR-155, which are integral to HIV replication, and immune activation. Using miRNA isolation and analysis, RNA interference, quantitative real time PCR, and reporter assays we explored the effects of cocaine on miR-155 and miR-20 in the context of HIV infection. Here we demonstrate using monocyte-derived dendritic cells (MDCCs) that cocaine significantly inhibited miR-155 and miR-20a expression in a dose dependent manner. Cocaine and HIV synergized to lower miR-155 and miR-20a in MDDCs by 90%. Cocaine treatment elevated LTR-mediated transcription and PU.1 levels in MDCCs. But in context of HIV infection, PU.1 was reduced in MDDCs regardless of cocaine presence. Cocaine increased DC-SIGN and and decreased CD83 expression in MDDC, respectively. Overall, we show that cocaine inhibited miR-155 and prevented maturation of MDDCs; potentially, resulting in increased susceptibility to HIV-1. Our findings could lead to the development of novel miRNA-based therapeutic strategies targeting HIV infected cocaine abusers.

The Immunomodulatory Role of Alcohol on HIV-Infected Monocyte-Derived Dendritic Cells

Alcohol is known to induce inflammation in the presence of the human immunodeficiency virus (HIV). In our previous studies, we revealed that alcohol induces cannabinoid receptors which play a role in the regulation of inflammatory cytokine production in monocyte-derived dendritic cells (MDDC). However, the ability of alcohol to alter MDDC function during HIV infection has not been clearly elucidated yet. To study the potential impact of alcohol on HIV-infected MDDC (confirmed by p24 ELISA), monocytes were isolated from commercially available buffy coats and cultured for 7 days with GM-CSF and IL-4. MDDC were infected with HIV- 1Ba-L and treated with different concentrations of alcohol (0.1% band 0.2%) for 4-7 days. MDDC phenotype, endocytosis, cytokine production, and ability to transmit HIV to T cells were analyzed. Uninfected CD4+ T cells were co-cultured for 7 days with either infected/treated MDDC or the supernatants from infected/treated MDDC. Inflammatory cytokine arrays were performed using supernatants from HIV-infected MDDC treated with alcohol. Results showed that HIV positive MDDC treated with alcohol had higher levels of infection compared to untreated HIV positive controls. CD4+ T cells exposed to HIV-infected MDDC acquired 100-fold higher levels of p24 compared to CD4+ T cells exposed to only supernatants. CD4+ T cells exposed to HIV-infected and alcohol-treated MDDC had higher levels of infection compared to controls. Cytokine array data show dysregulation of cytokine production by alcohol. In addition, MDDC phenotype and endocytic capacity were altered in the alcohol treated MDDC. Our results indicate a crucial role of MDDC in HIV transmission to T cells and provide insights into the inflammatory role alcohol exerts on dendritic cell function in the context of HIV infection. Supported by the National Institute on Alcohol Abuse and Alcoholism award R00AA021264, the National Institute on Drug Abuse award R01DA034547, and the Institute on NeuroImmune Pharmacology at FIU.

Characterization of plasmacytoid dendritic cells in the CD4C/HIV transgenic mouse model

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Regulates Dendritic Cells and Myeloid Derived Suppressor Cells Development in the Lymphoma Microenvironment

Calcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies.

We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.

In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.

Characterization of plasmacytoid dendritic cells in the CD4C/HIV transgenic mouse model

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

iNOS-producing inflammatory dendritic cells constitute the major infected cell type during the chronic Leishmania major infection phase of C57BL/6 resistant mice.

Leishmania major parasites reside and multiply in late endosomal compartments of host phagocytic cells. Immune control of Leishmania growth absolutely requires expression of inducible Nitric Oxide Synthase (iNOS/NOS2) and subsequent production of NO. Here, we show that CD11b+ CD11c+ Ly-6C+ MHC-II+ cells are the main iNOS-producing cells in the footpad lesion and in the draining lymph node of Leishmania major-infected C57BL/6 mice. These cells are phenotypically similar to iNOS-producing inflammatory DC (iNOS-DC) observed in the mouse models of Listeria monocytogenes and Brucella melitensis infection. The use of DsRed-expressing parasites demonstrated that these iNOS-producing cells are the major infected population in the lesions and the draining lymph nodes. Analysis of various genetically deficient mouse strains revealed the requirement of CCR2 expression for the recruitment of iNOS-DC in the draining lymph nodes, whereas their activation is strongly dependent on CD40, IL-12, IFN-gamma and MyD88 molecules with a partial contribution of TNF-alpha and TLR9. In contrast, STAT-6 deficiency enhanced iNOS-DC recruitment and activation in susceptible BALB/c mice, demonstrating a key role for IL-4 and IL-13 as negative regulators. Taken together, our results suggest that iNOS-DC represent a major class of Th1-regulated effector cell population and constitute the most frequent infected cell type during chronic Leishmania major infection phase of C57BL/6 resistant mice.

'Introducing Dendritic Cells as a Novel Immune-Inspired Algorithm for Anomaly Detection'

Abstract. Dendritic cells are antigen presenting cells that provide a vital link between the innate and adaptive immune system. Research into this family of cells has revealed that they perform the role of coordinating T-cell based immune responses, both reactive and for generating tolerance. We have derived an algorithm based on the functionality of these cells, and have used the signals and differentiation pathways to build a control mechanism for an artificial immune system. We present our algorithmic details in addition to some preliminary results, where the algorithm was applied for the purpose of anomaly detection. We hope that this algorithm will eventually become the key component within a large, distributed immune system, based on sound immunological concepts.

Introducting Dendritic Cells as a Novel Immune-Inspired Algorithm for Anomaly Detection

Dendritic cells are antigen presenting cells that provide a vital link between the innate and adaptive immune system. Research into this family of cells has revealed that they perform the role of coordinating T-cell based immune responses, both reactive and for generating tolerance. We have derived an algorithm based on the functionality of these cells, and have used the signals and differentiation pathways to build a control mechanism for an artificial immune system. We present our algorithmic details in addition to some preliminary results, where the algorithm was applied for the purpose of anomaly detection. We hope that this algorithm will eventually become the key component within a large, distributed immune system, based on sound imnological concepts.

Dendritic Cells for SYN Scan Detection

Artificial immune systems have previously been applied to the problem of intrusion detection. The aim of this research is to develop an intrusion detection system based on the function of Dendritic Cells (DCs). DCs are antigen presenting cells and key to the activation of the human immune system, behaviour which has been abstracted to form the Dendritic Cell Algorithm (DCA). In algorithmic terms, individual DCs perform multi-sensor data fusion, asynchronously correlating the fused data signals with a secondary data stream. Aggregate output of a population of cells is analysed and forms the basis of an anomaly detection system. In this paper the DCA is applied to the detection of outgoing port scans using TCP SYN packets. Results show that detection can be achieved with the DCA, yet some false positives can be encountered when simultaneously scanning and using other network services. Suggestions are made for using adaptive signals to alleviate this uncovered problem.

Dendritic Cells for Real-Time Anomaly Detection

Dendritic Cells (DCs) are innate immune system cells which have the power to activate or suppress the immune system. The behaviour of human DCs is abstracted to form an algorithm suitable for anomaly detection. We test this algorithm on the real-time problem of port scan detection. Our results show a significant difference in artificial DC behaviour for an outgoing portscan when compared to behaviour for normal processes.

Dendritic Cells for Anomaly Detection

Artificial immune systems, more specifically the negative selection algorithm, have previously been applied to intrusion detection. The aim of this research is to develop an intrusion detection system based on a novel concept in immunology, the Danger Theory. Dendritic Cells (DCs) are antigen presenting cells and key to the activation of the human immune system. DCs perform the vital role of combining signals from the host tissue and correlate these signals with proteins known as antigens. In algorithmic terms, individual DCs perform multi-sensor data fusion based on time-windows. The whole population of DCs asynchronously correlates the fused signals with a secondary data stream. The behaviour of human DCs is abstracted to form the DC Algorithm (DCA), which is implemented using an immune inspired framework, libtissue. This system is used to detect context switching for a basic machine learning dataset and to detect outgoing portscans in real-time. Experimental results show a significant difference between an outgoing portscan and normal traffic.

'Introducing Dendritic Cells as a Novel Immune-Inspired Algorithm for Anomaly Detection'

Abstract. Dendritic cells are antigen presenting cells that provide a vital link between the innate and adaptive immune system. Research into this family of cells has revealed that they perform the role of coordinating T-cell based immune responses, both reactive and for generating tolerance. We have derived an algorithm based on the functionality of these cells, and have used the signals and differentiation pathways to build a control mechanism for an artificial immune system. We present our algorithmic details in addition to some preliminary results, where the algorithm was applied for the purpose of anomaly detection. We hope that this algorithm will eventually become the key component within a large, distributed immune system, based on sound immunological concepts.