Extracellular mRNA induces dendritic cell activation by stimulating tumor necrosis factor-alpha secretion and signaling through a nucleotide receptor

We previously demonstrated that dendritic cell (DC) pulsing with antigen-encoded mRNA resulted in the loading of both major histocompatibility complex class I and II antigen presentation pathways and the delivery of an activation signal. Coculture of mRNA-pulsed DC with T cells led to the induction of a potent primary immune response. DC, in addition to recognizing foreign antigens through pattern recognition receptors, also must respond to altered self, transformed, or intracellularly infected cells. This occurs through cell surface receptors that recognize products of inflammation and cell death. In this report, we characterize two signaling pathways utilized by extracellular mRNA to activate DC. In addition, a novel ligand, poly(A), is identified that mediates signaling through a receptor that can be inhibited by pertussis toxin and suramin and can be desensitized by ATP and ADP, suggesting a P2Y type nucleotide receptor. The role of this signaling activity in vaccine design and the potential effect of mRNA released by damaged cells in the induction of immune responsiveness is discussed.     

RNA based vaccines

The recognition that CD8(+) T-cell mediated Th1 immune responses were necessary to produce immunity to intracellular and transformed self pathogens led to intense interest in the delivery of nucleic acids, DNA, or RNA encoding candidate antigens, as vaccines. Antigen presenting cells (APC) encounter most protein and vaccine immunogens as extracellular proteins and, thus, present them on major histocompatibility complex (MHC) class II molecules leading to the activation of CD4(+) T cells. Protein antigens encoded by nucleic acids delivered to dendritic cell (DC) are produced inside the cell and, thus, can stimulate MHC class I mediated activation of CD8(+) T-cell immune responses. Unfortunately, DCs are not readily transfected with DNA (Akbari et al., 1999) resulting in the requirement for high concentrations of DNA and repeated immunizations to achieved immune responses. RNA, on the other hand, is readily taken up and expressed by DC, making it an alternative vaccine candidate. In this article, we will discuss immune responses developed, interactions between APC and RNA that activate and dictate DC activation, and preliminary studies using RNA in vivo and in vitro to develop protective immunity.     

Early Activation of MAP Kinases by Influenza A Virus X-31 in Murine Macrophage Cell Lines

Early molecular responses to Influenza A (FLUA) virus strain A/X-31 H3N2 in macrophages were explored using J774.A1 and RAW 264.7 murine cell lines. NF-kappa B (NFκB) was reported to be central to FLUA host-response in other cell types. Our data showed that FLUA activation of the classical NFκB dependent pathway in these macrophages was minimal. Regulator proteins, IkappaB-alpha and –beta (IκBα, IκBβ), showed limited degradation peaking at 2 h post FLUA exposure and p65 was not observed to translocate from the cytoplasm to the nucleus. Additionally, the non-canonical NFκB pathway was not activated in response to FLUA. The cells did display early increases in TNFα and other inflammatory cytokine and chemokine production. Mitogen activated phosphokinase (MAPK) signaling pathways are also reported to control production of inflammatory cytokines in response to FLUA. The activation of the MAPKs, cJun kinases 1 and 2 (JNK 1/2), extracellular regulated kinases 1 and 2 (ERK 1/2), and p38 were investigated in both cell lines between 0.25 and 3 h post-infection. Each of these kinases showed increased phosphorylation post FLUA exposure. JNK phosphorylation occurred early while p38 phosphorylation appeared later. Phosphorylation of ERK 1/2 occurred earlier in J774.A1 cells compared to RAW 264.7 cells. Inhibition of MAPK activation resulted in decreased production of most FLUA responsive cytokines and chemokines in these cells. The results suggest that in these monocytic cells the MAPK pathways are important in the early response to FLUA.     

Noncompetitive Amine Uptake Inhibition by the New Antidepressant Pridefine

Abstract: Pridefine (AHR-1118) is a pyrrolidine derivative with clinically established antidepressant efficacy. Previous work from this laboratory indicates that pridefine is a reuptake blocker of catecholamines and serotonin with weak releasing activity. This study characterized the mode of amine uptake inhibition by pridefine as noncompetitive. The uptake experiments were performed utilizing ouabain instead of zero-degree controls to differentiate between the passive and active components of uptake. Furthermore, the passive component was resolved into diffusion and binding of substrate. Correction was made for the effects of ouabain on binding. Kinetic constants determined from Lineweaver-Burk plots were: K_m= 3 × 10^?7 M for NE, K_m= 9 × 10^?8 M for DA, and K_m= 3 × 10^?8 M for 5-HT. Dixon analyses of uptake at various pridefine concentrations indicated noncompetitive inhibition with K_i= 2.5 × 10^?6 M for NE uptake, K_i= 2.0 × 10^?6 M for DA uptake, and K_i= 1 × 10^?5 M for 5-HT uptake. These constants compare well with IC₅₀ values for the same transmitters: NE, IC₅₀= 2.4 × 10^?6 M; DA, IC₅₀= 2.8 × 10^?6 M; 5-HT, IC₅₀= 1.0 × 10^?5 M. The in vitro results indicate that pridefine is relatively specific as a catecholamine uptake blocker. It differs from tricyclic antidepressants which are reportedly competitive inhibitors of monoamine uptake. The possible mechanisms by which pridefine acts as a noncompetitive inhibitor are discussed.     

gp340 expressed on human genital epithelia binds HIV-1 envelope protein and facilitates viral transmission

During sexual transmission of HIV in women, the first cells likely to be infected are submucosal CD4(+) T cells and dendritic cells of the lower genital tract. HIV is segregated from these target cells by an epithelial cell layer that can be bypassed even when healthy and intact. To understand how HIV penetrates this barrier, we identified a host protein, gp340, that is expressed on genital epithelium and binds the HIV envelope via a specific protein-protein interaction. This binding allows otherwise subinfectious amounts of HIV to efficiently infect target cells and allows this infection to occur over a longer period of time after binding. Our findings suggest a mechanism of viral entry during heterosexual transmission where HIV is bound to intact genital epithelia, which then promotes the initial events of infection. Understanding this step in the initiation of infection will allow for the development of tools and methods for blocking HIV transmission.     

HIV envelope binding by macrophage-expressed gp340 promotes HIV-1 infection

The scavenger receptor cysteine-rich protein gp340 functions as part of the host innate immune defense system at mucosal surfaces. In the genital tract, its expression by cervical and vaginal epithelial cells promotes HIV trans-infection and may play a role in sexual transmission. Gp340 is an alternatively spliced product of the deleted in malignant brain tumors 1 (DMBT1) gene. In addition to its innate immune system activity, DMBT1 demonstrates instability in multiple types of cancer and plays a role in epithelial cell differentiation. We demonstrate that monocyte-derived macrophages express gp340 and that HIV-1 infection is decreased when envelope cannot bind it. Inhibition of infection occurred at the level of fusion of M-, T-, and dual-tropic envelopes. Additional HIV-1 envelope binding molecules, such as dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), mannose-binding lectin, and heparan sulfate, enhance the efficiency of infection of the cells that express them by increasing the local concentration of infectious virus. Our data suggest that gp340, which is expressed by macrophages in vivo, may function to enhance infection in much the same manner. Its expression on tissue macrophages and epithelial cells suggests important new opportunities for HIV-1 pathogenesis investigation and therapy.     

gp340 Promotes Transcytosis of Human Immunodeficiency Virus Type 1 in Genital Tract-Derived Cell Lines and Primary Endocervical Tissue

The human scavenger receptor gp340 has been identified as a binding protein for the human immunodeficiency virus type 1 (HIV-1) envelope that is expressed on the cell surface of female genital tract epithelial cells. This interaction allows such epithelial cells to efficiently transmit infective virus to susceptible targets and maintain viral infectivity for several days. Within the context of vaginal transmission, HIV must first traverse a normally protective mucosa containing a cell barrier to reach the underlying T cells and dendritic cells, which propagate and spread the infection. The mechanism by which HIV-1 can bypass an otherwise healthy cellular barrier remains an important area of study. Here, we demonstrate that genital tract-derived cell lines and primary human endocervical tissue can support direct transcytosis of cell-free virus from the apical to basolateral surfaces. Further, this transport of virus can be blocked through the addition of antibodies or peptides that directly block the interaction of gp340 with the HIV-1 envelope, if added prior to viral pulsing on the apical side of the cell or tissue barrier. Our data support a role for the previously described heparan sulfate moieties in mediating this transcytosis but add gp340 as an important facilitator of HIV-1 transcytosis across genital tract tissue. This study demonstrates that HIV-1 actively traverses the protective barriers of the human genital tract and presents a second mechanism whereby gp340 can promote heterosexual transmission.Through correlative studies with macaques challenged with simian immunodeficiency virus (SIV), the initial targets of infection in nontraumatic vaginal exposure to human immunodeficiency virus type 1 (HIV-1) have been identified as subepithelial T cells and dendritic cells (DCs) (18, 23, 31, 36-38). While human transmission may differ from macaque transmission, the existing models of human transmission remain controversial. For the virus to successfully reach its CD4⁺ targets, HIV must first traverse the columnar mucosal epithelial cell barrier of the endocervix or uterus or the stratified squamous barrier of the vagina or ectocervix, whose normal functions include protection of underlying tissue from pathogens. This portion of the human innate immune defense system represents a significant impediment to transmission. Studies have placed the natural transmission rate of HIV per sexual act between 0.005 and 0.3% (17, 45). Breaks in the epithelial barrier caused by secondary infection with other sexual transmitted diseases or the normal physical trauma often associated with vaginal intercourse represent one potential means for viral exposure to submucosal cells and have been shown to significantly increase transmission (reviewed in reference 11). However, studies of nontraumatic exposure to SIV in macaques demonstrate that these disruptions are not necessary for successful transmission to healthy females. This disparity indicates that multiple mechanisms by which HIV-1 can pass through mucosal epithelium might exist in vivo. Identifying these mechanisms represents an important obstacle to understanding and ultimately preventing HIV transmission.Several host cellular receptors, including DC-specific intercellular adhesion molecule-grabbing integrin, galactosyl ceramide, mannose receptor, langerin, heparan sulfate proteoglycans (HSPGs), and chondroitin sulfate proteoglycans, have been identified that facilitate disease progression through binding of HIV virions without being required for fusion and infection (2, 3, 12, 14, 16, 25, 29, 30, 43, 46, 50). These host accessory proteins act predominately through glycosylation-based interactions between HIV envelope (Env) and the host cellular receptors. These different host accessory factors can lead to increased infectivity in cis and trans or can serve to concentrate and expose virus at sites relevant to furthering its spread within the body. The direct transcytosis of cell-free virus through primary genital epithelial cells and the human endometrial carcinoma cell line HEC1A has been described (7, 9); this is, in part, mediated by HSPGs (7). Within the HSPG family, the syndecans have been previously shown to facilitate trans infection of HIV in vitro through binding of a specific region of Env that is moderately conserved (7, 8). This report also demonstrates that while HSPGs mediate a portion of the viral transcytosis that occurs in these two cell types, a significant portion of the observed transport occurs through an HSPG-independent mechanism. Other host cell factors likely provide alternatives to HSPGs for HIV-1 to use in subverting the mucosal epithelial barrier.gp340 is a member of the scavenger receptor cysteine-rich (SRCR) family of innate immune receptors. Its numerous splice variants can be found as a secreted component of human saliva (34, 41, 42) and as a membrane-associated receptor in a large number of epithelial cell lineages (22, 32, 40). Its normal cellular function includes immune surveillance of bacteria (4-6, 44), interaction with influenza A virus (19, 20, 32, 51) and surfactant proteins in the lung (20, 22, 33), and facilitating epithelial cell regeneration at sites of cellular inflammation and damage (27, 32). The secreted form of gp340, salivary agglutinin (SAG), was identified as a component of saliva that inhibits HIV-1 transmission in the oral pharynx through a specific interaction with the viral envelope protein that serves to agglutinate the virus and target it for degradation (34, 35, 41). Interestingly, SAG was demonstrated to form a direct protein-protein interaction with HIV Env (53, 54). Later, a cell surface-associated variant of SAG called gp340 was characterized as a binding partner for HIV-1 in the female genital tract that could facilitate virus transmission to susceptible targets of infection (47) and as a macrophage-expressed enhancer of infection (10).