HIV-1 is a sexually transmitted disease that primarily infects CD4+ T cells, dendritic cells and macrophages. Mucosal dendritic cells are among the first cells to encounter invading pathogens and it is becoming clear that different dendritic cells subsets have distinct functions in HIV-1 transmission. In the upper mucosal layers, Langerhans cells form a dense network that protects against HIV-1 infection . In mucosa and dermis, mucosal and dermal dendritic cells, respectively, can become infected by HIV-1 and are hijacked by HIV-1 to facilitate transmission to T cells [2,3]. Dendritic cells can activate HIV-1 specific immune responses, resulting in production of neutralizing antibodies and activation of cytotoxic T cells. Together, these immune reactions suppress viral replication after the primary viremia. However, HIV-1 has also been reported to suppress the immunogenicity of dendritic cells by preventing activation [4,5] and blocking autophagy [6•]. These studies show that dendritic cells are involved in different aspects of HIV-1 infection and the initiation of immune responses against HIV-1. Further studies on this subject will lead to better understanding of immune (dis)regulation during HIV-1 infection. This information is crucial to the development of better antiviral drugs and efficient vaccine strategies to stop the HIV-1 pandemic.
Dendritic cells bridge innate and adaptive immunity
Dendritic cells need to be able to discriminate between different classes of pathogens to induce proper adaptive immune responses. Dendritic cells recognize pathogens via pattern recognition receptors (PRR) that bind to pathogen-associated molecular patterns (PAMP). Dendritic cells express several classes of PRR, including toll-like receptors (TLR), nucleotide-binding oligomerization domain (NOD)-like receptors, retinoic acid inducible gene I (RIG-I)-like receptors and C-type lectin receptors (CLR) . These PRR induce signaling events that activate immune responses and tailor them to induce pathogen-specific responses. Therefore, the array of PRR that recognize a pathogen determines the type of adaptive immune response that is induced by dendritic cells. TLR and CLR recognize PAMP at the cell surface or after uptake in endosomes/lysosomes, and are therefore able to sense HIV-1 prior to infection of the cell. Cytosolic PAMP are recognized by sensors such as NOD- and RIG-I-like receptors or TRIM5, of which the latter has been recently shown to signal upon HIV-1 recognition [8••]. New insights have shown that these innate signaling pathways are not necessarily beneficial to the host. HIV-1 abuses their signaling pathways to decrease immune activation and dampen antiviral responses, enhance transmission to T cells, and increase HIV-1 replication.
The C-type lectin DC-SIGN is highly expressed by dendritic cells and interacts with the envelope protein of HIV-1, gp120, which leads to HIV-1 capture and viral transmission. Notably, it has become clear that DC-SIGN binding by pathogens such as HIV-1 also induces signals that shape adaptive immunity and/or promote HIV-1 infection and transmission. DC-SIGN recognizes both mannose- and fucose-expressing pathogens, which include bacteria, viruses and helminths. Recent data show that DC-SIGN is crucial in shaping adaptive immune responses to specific pathogens [9,10]. Notably, DC-SIGN triggering alone does neither result in dendritic cells maturation nor in transcription of inflammatory genes. However, signaling induced by DC-SIGN shapes immune responses by modifying signaling cascades activated by other PRR, which can enhance or suppress specific proinflammatory cytokines . Notably, signaling induced by DC-SIGN discriminates between mannose- and fucose-containing pathogens . Mannose-expressing pathogens such as HIV-1 require a signalosome that is preassembled at the cytosolic domain of DC-SIGN. This signalosome contains KSR1, CNK and Raf-1, that link to the cytoplasmic domain of DC-SIGN through the adaptor protein LSP-1. This signalosome is required for the constitutive recruitment of Raf-1 to DC-SIGN. Upon binding of DC-SIGN by HIV-1 and other mannose-expressing pathogens, Raf-1 becomes activated by recruitment of the upstream effectors LARG and RhoA to the DC-SIGN signalosome. Signaling downstream of Raf-1 induces phosphorylation of the NF-κB subunit p65 at serine 276, which allows subsequent acetylation of p65 at different lysine residues [9,10]. Acetylation of p65 prolongs its activity and also enhances its transcription rate of genes such as il12a, il12b and il6. Thus, p65 acetylation enhances expression of proinflammatory cytokines that are required for T helper cell type 1 differentiation . Noteworthy, Raf-1 activation alone does not induce NF-κB activation and prior triggering of another PRR such as TLR is required to activate NF-κB subunit p65. In contrast, fucose-expressing ligands actively disassociate KSR1, CNK and Raf-1 from DC-SIGN, which results in activation of a signaling cascade that suppresses proinflammatory cytokines . HIV-1 triggering of DC-SIGN activates Raf-1, which enhances TLR-induced proinflammatory cytokines IL-12p70 and IL-6 as well as the suppressive cytokine IL-10 . These data suggest that HIV-1 can use this modulatory signaling pathway to affect adaptive immune responses in HIV-1-infected individuals. Currently, it is unclear whether the modulation is beneficial to the host or virus.
DC-SIGN and Toll-like receptor-8 license HIV-1 transcription in dendritic cells
Dendritic cells capture HIV-1 through DC-SIGN, which facilitates transmission of HIV-1 to T cells . Internalization of HIV-1 by DC-SIGN leads to routing into endosomes, in which HIV-1 is protected from degradation. Therefore, dendritic cells have been postulated to play a pivotal role in HIV-1 transmission to T cells. Furthermore, recently it has been shown that DC-SIGN signaling is required for infection of dendritic cells. Uptake of HIV-1 by dendritic cells results in activation of p65 through triggering of TLR-8 by HIV-1 ssRNA [12••]. Induction of p65 results in recruitment of cyclin-dependent kinase (CDK)-7 toward the long terminal repeat (LTR), the promoter/enhancer of HIV-1. Notably, RNA polymerase II (RNAPII) is already present on the LTR without need for any signaling, demonstrating that LTR is a poised promoter. CDK-7 mediates phosphorylation of RNAPII at serine 2 of the C-terminal domain (CTD). This phosphorylation is required for initiation of transcription by RNAPII. However, transcription initiation does not lead to full-length HIV-1 transcripts and transcription will abort after about 65 bases without additional signals. Hence, TLR-8 triggering alone results in abortive HIV-1 transcription [12••]. The second signal, that leads to transcriptional elongation, is provided by binding of gp120 to DC-SIGN. The interaction of HIV-1 gp120 with DC-SIGN results in activation of Raf-1 and subsequently phosphorylation of p65 at serine 276. This modification recruits positive transcription elongation factor b (p-TEFb) to the LTR. p-TEFb phosphorylates the CTD of RNAPII at a serine 5 [12••], a hallmark for transcriptional elongation. Together, HIV-1-induced innate signaling by DC-SIGN and TLR-8 results in full-length transcription of the HIV-1 genome and production of HIV-1 proteins. The early gene product HIV-1 Tat is able to recruit p-TEFb to the LTR, which provides a positive feedback loop to sustain transcription of HIV-1 genome. Thus, HIV-1-induced innate signaling via TLR-8 and DC-SIGN controls transcription initiation and elongation from the integrated provirus and enables HIV-1 replication in dendritic cells. Coinfections, such as with fungi, also induce Raf-1 activation and thereby can further increase HIV-1 transcription [10,12••]. These studies show that HIV-1 subverts innate signaling mechanisms for infection of dendritic cells and subsequent transmission.
DC-SIGN ruffles the cell membrane to enhance HIV-1 transmission
Dendritic cells have been postulated to play a pivotal role in HIV-1 dissemination. HIV-1 transmission from dendritic cells to T cells is an efficient process that makes use of cell-to-cell transfer of the virus. At the dendritic cells–T-cell interface, actin-dependent membrane deformations form the infectious synapse (also called the virological synapse) , in analogy to what is observed for the immunological synapse. Binding of HIV-1 to dendritic cell-SIGN enhances infectious synapse formation [13,14], and it was recently shown that DC-SIGN signaling is involved in this process [15•]. Within an hour after HIV-1 exposure, the membrane starts to form extensions at the entire border of the cell. Exposure of dendritic cells to HIV-1 or DC-SIGN agonistic antibody H200, but not to HIV-1Δenv, leads to activation of Src kinases and downstream activation of Pak1, WASP and Cdc42. Activation of Cdc42 is required for formation of membrane extensions and transport of HIV-1 virions to these extensions. Cdc42 silencing reduces the amount of membrane extensions, and hence reduces the contact area with T cells and transmission of HIV-1. Moreover, a similar reduction of HIV-1 transmission was observed in LARG-silenced cells, indicating that LARG is also involved in membrane extension formation [15•]. LARG was previously shown to activate Rho-GTPases after binding of HIV-1 to DC-SIGN , suggesting that LARG activation is upstream of Cdc42 activation. Thus, HIV-1 subverts DC-SIGN signaling not only to induce transcription, but also to enhance the formation of the infectious synapse between dendritic cells and T cells and thereby facilitates its transmission to T cells. These signaling events by DC-SIGN might be important during HIV-1 transmission. However, in later stages of disease DC-SIGN might provide signals that reactivate HIV-1 replication and thereby keeps fueling transmission to T cells. Furthermore, shedding of viral gp120 during chronic infection might affect adaptive immunity induced by dendritic cells through triggering of specific signaling processes.
Dendritic cell immunoreceptor increases HIV-1 susceptibility
Another C-type lectin that is expressed by dendritic cells and is involved in recognition of HIV-1 is dendritic cell immunoreceptor (DCIR) . Similar to DC-SIGN, DCIR facilitates transmission of HIV-1 from dendritic cells to T cells and enhances infection of DCIR expressing cells . Recently, it was shown that DCIR-induced signaling results in increased HIV-1 entry/binding [19•]. The DCIR signaling pathway involves the ITIM motive present in its cytosolic domain. Competitive peptides containing the ITIM-domain reduced HIV-1 capture and infection of dendritic cells. Ligation of DCIR results in recruitment of Src homology 2-containing tyrosine phosphatase (SHP)-1 and SHP-2. Enhancement of HIV-1 binding/entry is dependent on Src family members Src, Fyn and Hck; the Syk family; and PKC-α [19•]. It has to be established whether signaling of DCIR leads to enhance capture or that it enhances internalization. DCIR signaling could also be involved in recruitment of CD4 and/or the HIV-1 coreceptors to HIV-1 because its signaling is required for the enhanced infection seen in cells transfected with DCIR. Thus, DCIR is a novel receptor that HIV-1 abuses to promote its transmission and infection.
HIV-1 capsid recognition increases antiviral responses
Cytosolic recognition of HIV-1 also plays an important role in HIV-1 infection. TRIM proteins are cytosolic innate immune sensors that are known to restrict virus infections. TRIM5 can recognize the lattice formed by HIV-1 capsid protein upon infection [8••]. TRIM5 accelerates the uncoating of the core, which negatively influences HIV-1 infectivity . Besides this direct effect on HIV-1, TRIM5 promotes innate signaling, which is enhanced after HIV-1 infection [8••]. Upon capsid recognition, TRIM5 induces transcriptional activation of NF-κB and AP1, and enhances IRF3-mediated interferon (IFN)-β transcription. TRIM5 acts with CBC13 and UEV1A to form free lysine-63-linked polyubiquitin chains that activate TAK-1. Subsequently, downstream of TAK-1 the transcription factors AP1 and NF-κB are activated. Hence, TRIM5 is a PRR that recognizes HIV-1 infection before integration of HIV-1 into the host genome. This results in a two-pronged response, leading to degradation of the viral core and activation of the immune system.
HIV-1 capsid protein is also recognized at another stage of the HIV-1 live cycle. De novo synthesized capsid can be recognized by the cytosolic receptor cyclophilin A [21••]. Upon recognition of capsid by cyclophilin A, dendritic cells mature, produce type-1 IFN and induce HIV-1-specific CD4+ and CD8+ T cells [21••]. Type-1 IFNs are well known for their antiviral properties and this results in an antiviral state that suppresses transmission to, or infection of T cells. However, in these experiments dendritic cells were coinfected with a simian immunodeficiency virus (SIV)-like particle expressing SIV vpx to overcome limited infection of dendritic cells by HIV-1. The latter is debated in literature, as other groups have reported infection of dendritic cells with HIV-1 [2,12••,19•]. Thus, further research is needed to address the differences between the efficiency of HIV-1 infection of dendritic cells in different laboratories, and to the effect of SIV coinfections on dendritic cells.
HIV-1 exhausts autophagy and thereby limits dendritic cell activation and induction of T cell responses
Dendritic cells can degrade cytosolic antigens by a specialized lysosomal degradation pathway called autophagy [22,23]. This pathway is mainly used for self-digestion. However, it has also been shown to be involved in the induction of immune responses against cytosolic pathogens [24,25] by targeting them to stimulate TLR . Therefore, autophagy is a powerful tool for dendritic cells to induce anti-HIV-1 immune responses. Indeed, HIV-1 ends up in lysosomes after autophagocytosis [6•]. However, HIV-1 shuts autophagy down by activation of mTOR signaling [6•]. Down-regulation of autophagy by HIV-1 leads to increased HIV-1 levels in dendritic cells and increased transmission to T cells. Similarly, enhancing autophagy by silencing negative regulators or stimulation with rapamycin boosts HIV-1 degradation and HIV-1-antigen presentation to specific T cells. Besides protecting HIV-1 from autophagocytosis, this pathway probably prevents immune activation because HIV-1-induced downregulation of autophagy reduces the sensitivity of dendritic cells to TLR ligands. This shows that HIV-1 interferes with innate signaling pathways that are in place to promote antigen presentation and immune activation.
Tipping the balance of innate signaling
Type-I IFN production is associated with an antiviral state that suppresses viral replication and in-vitro, type-I IFN is able to reduce HIV-1 replication. Plasmacytoid dendritic cells have an innate capacity to produce large amounts of type-I IFN. Cell-free HIV-1  and HIV-1-infected cells  induce IFN production by plasmacytoid dendritic cells, which can suppress HIV-1 replication. The induction of IFN was linked to innate signaling events of TLR-7 and -9 and subsequent IRF3 transcription . However, excesses of IFN can induce chronic hyper activation of the immune system, which is one of the pathogenic effects in HIV . This suggests that although innate immune responses are highly potent in suppressing HIV-1 replication, repetitive induction of these signaling pathways can do more harm than good.
Not only HIV-1 itself induces innate signals that influence HIV-1 infection, but also coinfections induce innate responses that can interfere with HIV-1 infection. Langerhans cells are a subset of dendritic cells that protect against HIV-1 infection by degrading virions and prevent further spreading of the virus [1,29]. However, also Langerhans cells can be infected by HIV-1 during inflammation [30–32]. The precise mechanisms still have to be deciphered, but it is evident that activation of Langerhans cells breaches their innate tolerance to HIV-1 infection and transmission. Moreover, also nonmucosal coinfection with Mycobacterium tuberculosis and Candida albicans has been shown to increase HIV-1 replication [33–35]. This suggests that HIV-1 benefits from immune activation, which might negatively influence disease progression. Therefore, the innate signals triggered by coinfections can make way for HIV-1 dissemination.
The innate immune system is highly potent in preventing spreading of pathogens, and innate signaling is key in this response. Several studies indicate that HIV-1 circumvents the same innate mechanisms by shutting them down. Strikingly, HIV-1 is able to use the receptors and/or their signaling pathways to enhance its infectivity. Therefore, detailed knowledge of these innate events that allows us to interfere where needed is essential in limiting HIV-1 replication. Most anti-HIV-1 drugs target viral proteins, but these new insights might provide targets that affect host processes to increase the immune response, whereas decreasing the susceptibility to HIV-1.
The authors are supported by the Netherlands Organisation for Scientific Research (NWO Vici 91810619, T.B.H.G.) and the AIDS Foundation (M.v.d.V: 2007036; A.M.G.v.d.A: 2009024).
Conflicts of interest
There are no conflicts of interest.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
* • of special interest
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