The human immunodeficiency virus is a double stranded RNA virus belonging to the lentivirus genus and the Retroviridae family. The distinguishing characteristic of HIV is that the capsid of the virus contains two identical single RNA strands. Upon entry of the target cell the single-stranded RNA is reverse transcribed by the viral RNA-dependent DNA polymerase, also referred to as the reverse transcriptase1. Two distinct sub-types of HIV, HIV-1 and HIV-2, are identified based on their introduction into human population as well as by their differential virulence to humans. The HIV type 1, HIV-1, is the descendent of chimpanzee derived Siminan Immunodeficiency Virus (SIVcpz), and it is the more virulent of the two form of HIV. HIV-1 accounts for the majority of infections worldwide (x,x). The HIV type 2, HIV-2, is a descendent of SIV isolated from sooty mangabey monkeys (SIVx), and is generally found in areas of West Africa. HIV-2 is less virulent and less transmissible compared to HIV-1 (x). HIV-2 was also distinguished from HIV-1 based on the longer clinical latency period and lower rate of morbidity of the resulting disease. Nevertheless, infection by either HIV-1 or HIV-2 ultimately leads to the development of AIDS in humans.
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The HIV-1 infection process begins with the binding of the viral envelope surface protein gp120 to the CD4 receptor on the target cell (Fig. 1.1). Once this binding occurs, the gp120 glycoprotein undergoes a series of conformational changes required for binding to a chemokine receptor, termed co-receptor 2. Different strains of HIV-1 are classified based upon their usage of the chemokine co-receptor: the T-cell-tropic viruses use the CXCR4 chemokine receptor and are referred to as X4 viruses 3 while the macrophage-tropic viruses use the CCR5 chemokine receptor and are referred to as R5 viruses 3, 4-5. Strains of HIV-1 that can utilize both types of chemokine co-receptors are referred to as dual-tropic viruses. It is generally believed that HIV-1 transmissions in humans are initiated with the infection by the macrophage-tropic HIV-1 strains and during progressive infection, the virus shifts toward gaining T-cell tropism. This shift in viral tropism usually occurs late in the course of disease and marks an accelerated phase in the clinical disease progression (x). Once the gp120-CD4 complex is bound to the appropriate co-receptor, additional conformational changes expose the transmembrane protein gp41 that gets inserted into the membrane of the target cell for subsequent fusion of the viral and target cell membranes (Fig. 1.1). One consequence of HIV infection is the decline in the numbers of total and different memory subsets of CD4+ T cells and when the decline reaches below a critical level, HIV infected individuals develop susceptibility to opportunistic infections that eventually culminate into the acquired immunodeficiency syndrome (AIDS). In 2007, there were an estimated 33.2 million cases of HIV infection worldwide with oral and genital mucosal infections accounting for the majority of all reported cases of HIV-1 infection and transmission 6.
Fig. 1.1: Cartoon representing the process of HIV-1 entry into human cells: The surface envelope glycoprotein gp120 of the virus binds to the CD4 receptor on the host cell membrane resulting in a conformational change in gp120 (a) that enables co-receptor binding (b) followed by the exposure of the gp41 transmembrane glycoprotein (c) for fusion of the viral and cell membranes (d).
Source: Doranz et al. (x).
The rhesus macaque (Macaca mulatta) model is widely used to extensively study HIV transmission. This model uses either the simian immunodeficiency virus (SIV), a primate lentivirus closely related to HIV, or a laboratory-created hybrid virus called simian-human immunodeficiency virus (SHIV) consisting of genes that encode the HIV envelope and the SIV core. Both of these viruses can be easily transmitted to macaques by vaginal inoculation and cause AIDS-like illness characterized by severe loss of CD4+ T cells and opportunistic infections. This model of SIV/SHIV infection of macaques is similar to that of HIV in humans in that the target cells, physiology, and immunology of the genital tract are comparable. Using this model, Miller et. al described the propagation and dissemination of SIV after vaginal transmission7 (Fig. 1.2). Their study showed that between days 1-4 post-infection only a few infected cells could be found in the vaginal tissue, the original site of inoculation, as well as at distant gut-associated lymphoid tissues (GALT). However, beyond day 4 post-infection, there is a substantial increase in infected cells at the local vaginal tissue and a concurrent increase over days 10-14 at the GALT. These observations correlate well with later reports describing mucosal SIV infection in rhesus macaques and HIV infection in humans resulting in a rapid depletion of intestinal CD4+ T cells by day 14 post-exposure. This extreme level of depletion of CD4+ T cells occurs primarily in the mucosal tissues of the gastrointestinal (GI) tract where more than 60% of T lymphocytes reside 8-12. It is unclear how the virus effectively manipulates the mucosal microenvironment leading to the systemic spread of infection since the role the mucosal epithelium plays in sensing HIV infection, triggering immune activation, and promoting HIV spread and amplification remains unknown. A better understanding of the detailed steps during the mucosal infection process should provide a framework for understanding the propagation, dissemination, and establishment of infection during the acute stage of infection.
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Fig. 1.2: Model of delayed systemic SIV replication. Study from Miller et al. showing that between days 1-4 post-infection only a few infected cells could be found in the vaginal tissue, the original site of inoculation, as well as at distant lymphoid tissues. However, beyond day 4 post-infection, there is a substantial increase in infected cells at the local vaginal tissue and a concurrent increase over days 10-14 at the distant lymphoid tissues.
Source: Miller et al. (ref) The Mucosal Microenvironment
About 30-40% of all new HIV cases occur in women through vaginal intercourse, a route that carries the lowest HIV transmission probability per exposure suggesting that the genital epithelium fails to serve as a major barrier against HIV entry. For successful virus transmission, HIV must be efficient in devising strategies to cross the mucosal barrier of the genital tract to infect CD4+T cells. Since oral and genital mucosal epithelial cells express low to negligible levels of the receptors for HIV, in particular CD4, the virus has to utilize unconventional mechanisms to cross primary genital epithelial cell layers. Studies in the rhesus macaque model with SIV suggested that the initial targets of infection may be Langerhans Cells (LCs), subepithelial T-cells and dendritic cells (DCs) (x,x,x,x,x). In order for the virus to reach these targets it must successfully penetrate the epithelial barrier. There are several proposed mechanisms for how HIV-1 may cross the mucosal epithelium (Fig. 1.3). The susceptibility of epithelial cells to HIV infection is controversial with some studies reporting that different epithelial cell lines can be productively infected and transfer virus to gut-associated epithelial cells based on the expression of certain glycosphingolipids such as galactosylceramide (GalCer). Other studies have suggested that genital epithelial cells can bind, transport, harbor, and transmit virus to additional targets, but cannot be productively infected (x, x, x) 13-17. A second proposed mechanism for viral entry across the mucosal epithelium is by transmigration of infected cells through the mucosal epithelial cells. These cells can include infected CD4+ T cells and monocytes found in semen (x,x). Langerhans cells (LC) constitute another vehicle by which virus may traverse the mucosal epithelium because it has been shown that LC can sample the space beyond the mucosal barrier for foreign antigens. Since LC express CD4, CCR5, and CXCR4 and can be found within the genital tract it has been suggested that LC at these mucosal sites can capture HIV and migrate to the draining lymph nodes where the virus is transmitted to CD4+ T cells, the main targets of HIV replication and dissemination 21-22. Whether or not the LC become infected after exposure to virus inoculation is still controversial. Some studies of vaginal mucosal transmission of SIV in rhesus macaques demonstrated that submucosal LCs rapidly became infected after virus inoculation while other studies suggest that the CD4+ T cells are the cells infected and that they are the major source of infectious virus during the acute stages of infection. Ex vivo studies have demonstrated that LCs are not required for, but may aid in, viral transmission (x). A fourth mechanism by which HIV can be transmitted across the mucosal epithelium is through breaches in the epithelial layer. These breaches can occur as a result of trauma associated with sexual intercourse or as a result of infection with other sexually transmitted diseases that can compromise the strength of the mucosal barrier (x,x). These breaks in the epithelial barrier allow the virus access to susceptible targets of infection in the underlying tissues but studies in the rhesus macaque model have revealed that barrier disruption is not necessary for successfully viral transmission (x). The fifth proposed mechanism by which HIV can be transmitted across the mucosal epithelium is by transcytosis / endocytosis through the mucosal epithelium involving interactions with cell surface heparin sulfate moieties, enabling infection of the nearby dendritic cells (DC) and CD4+ T cells 17-20. This pathway was initially proposed after virus was observed being transported into endosome like compartments after coming into contact with the surface of epithelial cells (x). Ex vivo studies using the cervical explants model have also shown that HIV-1 virions can be found in endocytic compartments as well as in the cytosol of epithelial cells (x). Elimination of heparin sulfate moieties was not shown to abrogate viral transcytosis suggesting that other host cell factors may also be involved in viral transcytosis in epithelial cells (x). Several recent reports (refs) have demonstrated a specific interaction of HIV with epithelial cells through the binding of the HIV surface envelope protein gp120 to the cell surface salivary agglutinin (SAG) protein gp340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily of proteins and protein splice variant of the Deleted in Malignant Brain Tumors I (DMBT1) that can be either secreted or membrane-bound which may be involved in transcytosis of HIV.
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Potential routes of HIV transmission: a) Direct infection of epithelial cells. b) Transcytosis through epithelial cells. c) Transmigration of infected donor cells. d) Uptake by migratory intra-epithelial Langerhans cells. e) Direct entry through breaches in the epithelial layer.
Source: Shattock & Moore 2003 (ref)
Glycoprotein-340 (gp340) is a human protein splice variant of the Deleted in Malignant Brain Tumors I (DMBT1). DMBT1 has been identified as a member of the scavenger receptor cysteine-rich (SRCR) family of proteins, which are known for their role as pattern recognition receptors for interactions with specific pathogen motifs that drive signaling events (refs). Differentially spliced variants of DMBT1 were reported to be both as secreted forms or as membrane associated proteins (refs). Abundant expression has been found in tissues such as the lung, trachea, salivary glands, stomach and small intestine while minor expression has been found in the testis, mammary glands, uterus and brain (x). The gp340 protein, a cell membrane associated splice variant of DMBT1 has been found to be expressed at high levels in the lung and upon recognition of specific pathogen motifs has been shown to stimulate the migration of alveolar macrophages (refs). The gp340 protein, as the salivary agglutinin (SAG) protein , is the secreted splice variant of DMBT1 found in saliva and was initially identified based on its role in the clearance of bacterial pathogens including S. mutans by initiating clearance through agglutination and opsiniztion (refs). SAG was the first DMBT1 splice variant to be identified to play a role in HIV infection (refs). Compared to vaginal transmission, HIV transmission within the oral cavity is low despite the presence of similar cell types in both oral and vaginal tissues. Fractionation of human saliva led to the identification of SAG as an inhibitor of HIV infection in the oral cavity (refs). It was shown that SAG interaction with HIV leads to viral agglutination (x). Immunoprecipitation assays showed that SAG binds to gp120 in an area different from the CD4 binding site, and it was reported that pretreatment of HIV with soluble CD4 resulted in enhanced SAG-gp120 binding (x,x,x). Further studies revealed that SAG interacts with HIV within the base of the V3 loop, an area that is well conserved within the viral genome (x,x and Fig. 1.4). While the role of SAG in HIV biology has been studied in great detail, the role of epithelial cell surface gp340 in HIV transmission across genital mucosa has only recently begun to be characterized. Stoddard et al reported that gp340 is expressed on vaginal and cervical tissues and that the cells expressing the cell surface associated form of gp340 are capable of binding and facilitating HIV transmission. Since HIV is capable of interacting with epithelial cells surface gp340, it is possible that this receptor may play a role in the way mucosal epithelial cells sense viral antigens and trigger immune activation.
Fig.1.4: Model depicting interaction of gp340 SRCR domain with gp120: a) Model of the quatinary complex between gp120, CD4, AB17b, and the SRCR domain on gp340. b) Space filling model of interaction between gp120 and SRCR domain on gp340.
Source: Wu et al 2004 (ref)
B.3. Thymic Stromal Lymphopoietin (TSLP)
It is known that mucosal epithelial cells secrete thymic stromal lymphopoietin (TSLP) in response to allergens and bacteria (refs). TSLP is an IL-7 like cytokine that was originally identified in the supernatants of the mouse thymic stromal cell line, Z210R.1 and was reported to display a remarkable ability to support the long-term growth of a pre-B cell line as well as upregulate the proliferation of thymocytes (ref). Isolation of a cDNA clone encoding human TSLP revealed that it is structurally similar to murine TSLP but shares only 43% amino acid homology. The TSLP receptor complex is a heterodimer consisting of a TSLP receptor (TSLPR) binding chain and the interleukin 7 receptor-Î± (IL-7RÎ±) chain, together bind TSLP with a high affinity resulting in Signal Transducers and Activator of Transcription-5 (STAT-5) activation and cell proliferation23-26. Early studies demonstrated that unlike murine TSLP, the human TSLP does not promote the differentiation and growth of B cells nor does it have a direct effect to support the activation of T cells (refs).
In humans, TSLP is mainly expressed by skin keratinocytes, epithelial cells, smooth muscle cells, lung fibroblasts, or IgE stimulated mast cells. Microbial infection or allergen exposure triggers mucosal epithelial cells to produce TSLP23 (Fig.1.5). TSLP strongly up-regulates activation markers such as CD54, CD80, CD83, CD86, DC-SIGN, and HLA-DR on myeloid dendritic cells (mDC) and activates immature DC to produce the neutrophil and eosinophil attractant chemokines Interleukin-8 (IL-8) and eotaxin-2 as well as the T helper 2 (Th2) attractant chemokines thymus and activation regulated chemokine (TARC or CCL17) and macrophage-derived chemokine (MDC or CCL22). Unlike the DC activated by a variety of factors, such as CD40-L, LPS and poly I:C , TSLP-activated DC do not produce IL-12, TH1 responses, or the proinflammatory cytokines TNF, IL-1Î², and IL-6 (ref). Instead, mature TSLP-activated DC migrate to the draining lymph node and express high levels of OX40L, which triggers the homeostatic proliferation and differentiation of naÃ¯ve CD4+ T cells into a unique type of inflammatory Th2 cells that produce Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-13 (IL-13), and Tumor Necrosis Factor (TNF) and not the classic Th2 cells that produce Interleukin-10 (IL-10) in the place of TNF. These inflammatory Th2 cells then migrate back to the site of inflammation due to the local production of TARC and MDC. These studies suggest that TSLP produced by epithelial cells plays an important role in inducing allergic inflammatory responses. This hypothesis is supported by studies that show that the airway epithelium of asthma patients expresses increased levels of TSLP (25) and that infection by rhinoviruses or exposure to proinflammatory mediators such as IL-1Î², TNF-Î± and some TLR agonists induce TSLP production in human airway epithelial cells causing the exacerbation of asthma (26). Furthermore, TSLP is highly expressed in patients with acute and chronic atopic dermatitis lesions (ref) and this increased TSLP expression is associated with Langerhan cell migration and activation (Ito et al JEM 2005) suggesting that TSLP is a critical link between epithelial cells and the DC of the immune system at a molecular level. The fact that TSLP produced by epithelial cells exposed to allergens and microbes induces DC-mediated expansion of CD4+ T cells formed the basis for the central hypothesis of this dissertation research that: HIV exposure at the mucosal tissues stimulates epithelial cells to produce TSLP for DC-mediated expansion of susceptible target CD4 T cells.
In 2007, Lee and Ziegler identified an NFÎºB site within the human TSLP gene promoter that was critical for IL-1Î² and TNF-Î± induced TSLP expression. This provided a link for the mechanism by which TSLP expression is induced by ligands for TLR2, TLR8, and TLR9. Since NFÎºB has such a broad range of activation signals, several labs explored the potential involvement of other factors for regulating TSLP expression. Li et al. identified two nuclear receptor binding sites in both the human and mouse TSLP gene promoters, in particular the retinoid X receptors (RXR) Î± and Î² for which 9-cis- retinoic acid (9-cis-RA) serves as a high-affinity ligand (ref). Studies showed that keratinocytes with selective ablation of RXRÎ± and RXRÎ² display an inflammatory response similar to that of atopic dermatitis in humans. (ref). High levels of TSLP expression were also found in these cells. In 2008, Lee et al. demonstrated that 9-cis-RA represses IL-1Î² mediated TSLP gene expression through direct inhibition of NFÎºB binding and signaling. These studies suggest that RXRÎ± and RXRÎ², when bound to ligand, are involved in regulating TSLP expression and, combined with my results from the investigation, could offer new insights into developing novel therapeutic strategies in the fight against the transmission of HIV.
Fig.1.5: Pathology of TSLP in allergic inflammation
Microbial infection or allergen exposure triggers mucosal epithelial cells to produce TSLP. TSLP activates immature DC to produce the neutrophil and eosinophil attractant chemokines Interleukin-8 (IL-8) and eotaxin-2 as well as the T helper 2 (Th2) attractant chemokines thymus and activation regulated chemokine (TARC or CCL17) and macrophage-derived chemokine (MDC or CCL22). Matured TSLP-activated DC migrate to the draining lymph node and express high levels of OX40L, which triggers homeostatic proliferation and differentiation of naÃ¯ve CD4+ T cells into inflammatory Th2 cells that produce Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-13 (IL-13), and Tumor Necrosis Factor (TNF). These inflammatory Th2 cells then migrate back to the site of inflammation due to the local production of TARC and MDC. .
Source: Y.J. Liu, 2006 (ref)