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TNF-α and TLR agonists increase susceptibility to HIV-1 transmission by human Langerhans cells ex vivo
  Published September 5, 2008
J. Clin. Invest. doi:10.1172/JCI34721.
Marein A.W.P. de Jong1, Lot de Witte1, Menno J. Oudhoff2, Sonja I. Gringhuis1, Philippe Gallay3 and Teunis B.H. Geijtenbeek1
1Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands.
2Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Medical Center, Amsterdam, The Netherlands.
3Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA.
Genital coinfections increase an individual's risk of becoming infected with HIV-1 by sexual contact. Several mechanisms have been proposed to explain this, such as the presence of ulceration and bleeding caused by the coinfecting pathogen. Here we demonstrate that Langerhans cells (LCs) are involved in the increased susceptibility to HIV-1 in the presence of genital coinfections. Although LCs are a target for HIV-1 infection in genital tissues, we found that immature LCs did not efficiently mediate HIV-1 transmission in an ex vivo human skin explant model. However, the inflammatory stimuli TNF-α and Pam3CysSerLys4 (Pam3CSK4), the ligand for the TLR1/TLR2 heterodimer, strongly increased HIV-1 transmission by LCs through distinct mechanisms. TNF-α enhanced transmission by increasing HIV-1 replication in LCs, whereas Pam3CSK4 acted by increasing LC capture of HIV-1 and subsequent trans-infection of T cells. Genital infections such as Candida albicans and Neisseria gonorrhea not only triggered TLRs but also induced TNF-α production in vaginal and skin explants. Thus, during coinfection, LCs could be directly activated by pathogenic structures and indirectly activated by inflammatory factors, thereby increasing the risk of acquiring HIV-1. Our data demonstrate a decisive role for LCs in HIV-1 transmission during genital coinfections and suggest antiinflammatory therapies as potential strategies to prevent HIV-1 transmission.

HIV-1 infection, the causative agent of AIDS, is still on the rise. It was estimated that 2 to 4 million people acquired HIV-1 in 2007, while already 31 to 36 million people are HIV-1 infected worldwide (1). Thus, the HIV-1 pandemic, for which there is currently no cure or vaccine available, is still growing. Heterosexual transmission of HIV-1 across genital epithelial tissue is the primary route of HIV-1 dissemination worldwide (1, 2). Therefore, increased knowledge about how sexual transmission of HIV-1 occurs and the identification of factors facilitating or enhancing this process are essential for the development of effective strategies to reduce new infections.
Many biological factors are involved in sexual transmission of HIV-1, and the risk of HIV-1 acquisition varies depending on these factors, including viral loads (3), viral variants, and host susceptibility, which may include contraception, male circumcision, and genital coinfection (2). Moreover, different soluble factors in body fluids might influence transmission such as factors in breast milk (4) and semen (5). Furthermore, genital coinfections have been linked to increased susceptibility to HIV-1 and include ulcerative sexually transmitted diseases (STDs) such as genital herpes, gonorrhea, syphilis, and chlamydial infections (6) as well as yeast and bacterial vaginal infections (7-9). However, the mechanisms accounting for increased HIV-1 susceptibility in the presence of genital coinfections are unclear. It is argued that these infections increase susceptibility by recruiting HIV-1 target cells into the site of infection (10) or by causing ulceration and subsequent bleeding (6). In addition, we hypothesize that pathogenic structures or inflammatory cytokines that are induced upon infection change the function of key players in the transmission of HIV-1 (11).
Langerhans cells (LCs) are a subset of DCs that reside in the epidermis of skin and in mucosal epithelia such as ectocervix, vagina, and foreskin (12, 13). LCs are therefore likely to be the first cells that encounter HIV-1 upon sexual transmission. However, there is debate about whether LCs are also the first cells infected by HIV-1 and whether they are involved in the initiation of systemic disease (12, 14-16). Several ex vivo skin explant studies have shown that LCs are susceptible to HIV-1 and transmit HIV-1 to T cells (12, 17, 18). We have recently demonstrated that HIV-1 infection of LCs and subsequent transmission to T cells is an inefficient process. Langerin, a C-type lectin specifically expressed by LCs, captures HIV-1 and acts as a protective barrier for HIV-1 infection by targeting HIV-1 to Birbeck granules for degradation. However, when the Langerin function is blocked or saturated using high virus concentration this barrier can be overcome. These conditions allow LC infection and subsequent HIV-1 transmission to occur (16). Thus, LCs are an essential checkpoint where it is decided whether the virus is degraded or transmitted, and we hypothesize that its activation state and the encountered viral loads are a decisive factor.
Both inflammatory cytokines and pathogen-associated molecular patterns (PAMPs) induce LC activation (19-21), and these factors, which are present during coinfections, might breach the protective function of LCs to allow HIV-1 transmission. PAMPs are recognized by TLRs, and TLR triggering on LCs results in LC maturation (21). Here we set out to investigate the effect of bacterial and fungal coinfections on HIV-1 transmission by LCs. To mimic the epithelial environment, we developed an ex vivo skin explant transmission model to investigate the effect of coinfection on HIV-1 transmission. We demonstrate that TLR agonists, bacterial and fungal pathogens, induce the production of the proinflammatory cytokine TNF-α. Strikingly, both TNF-α and the TLR1/TLR2 ligand Pam3CysSerLys4 (Pam3CSK4) strongly enhance HIV-1 transmission by LCs using distinct mechanisms. TNF-α increases HIV-1 replication in LCs, whereas Pam3CSK4 enhances HIV-1 capture. Our data demonstrate that in response to inflammatory cytokines and pathogenic structures present during genital coinfection, LCs mediate HIV-1 transmission. Identification of these risk factors that increase HIV-1 susceptibility forces a reevaluation of LC function in HIV-1 transmission and might help toward the development of strategies to prevent HIV-1 transmission.
In order to reduce the growing HIV-1 pandemic, there is a strong pressure to understand HIV-1 transmission. Sexual transmission of HIV-1 is the major route of infection worldwide (1), and little is known about the factors governing HIV-1 transmission and susceptibility to HIV-1. Genital coinfection is a risk factor for acquiring HIV-1 (2), indicating that pathogens or host responses to the pathogens create a genital environment that favors HIV-1 transmission. Here we demonstrate a potential role for LCs in the increased susceptibility to HIV-1 during fungal and bacterial coinfections.
Due to their epithelial localization and function, LCs are the first cells to encounter HIV-1 in the genital epithelial tissues (12, 13). Several studies suggest that HIV-1 subverts the function of LCs for viral transmission to T cells, thereby infecting the host (18, 34). However, other reports argue that not LCs but T cells and subepithelial antigen-presenting cells are the first cells infected by HIV-1 (14, 15, 39). Moreover, our recent data demonstrate that LCs are not efficiently infected by HIV-1 and form a barrier to HIV-1 infection through the function of the C-type lectin Langerin (16); Langerin captures HIV-1, resulting in efficient virus degradation, which prevents infection. However, Langerin inhibitors or high virus concentrations (>100 ng p24/106 LCs) that saturate Langerin allow LC infection and subsequent HIV-1 transmission (16). These data suggest that under normal conditions LCs function as a barrier but that specific conditions might change LC function and promote transmission. Using an ex vivo transmission model, we demonstrate here that indeed HIV-1 transmission by LCs is low and inefficient; high concentrations of HIV-1 (30 ng p24 per epidermal sheets, containing approximately 105 LCs) were needed to observe transmission by LCs. Strikingly, activation of LCs by TNF-α or Pam3CSK4 induces a strong increase in transmission of HIV-1, suggesting that under conditions of high viral loads and inflammation, LCs play a pivotal role in HIV-1 transmission.
Inflammatory conditions induce LC maturation, which includes downregulation of antigen capture and processing as well as increased migration from the periphery to the lymphoid tissues. Monocyte-derived LC-like cells are more susceptible to HIV-1 infection after activation with CD40L (40). Moreover, a recent study demonstrated that CD34+-derived LC-like cells only efficiently transfer HIV-1 after activation by a combination of LPS and TNF-α (41). Although primary LCs are unresponsive to LPS (21), these studies using LC-like models suggest that LC activation is an important determinant in whether LCs protect against HIV-1 infection or transmit the virus to T cells. Moreover, the cellular environment might also be pivotal to LC function in HIV-1 transmission. Therefore, we have used an ex vivo skin model to investigate the role of primary LCs and the environment in HIV-1 transmission during coinfections and inflammatory conditions. Although the LC fractions contained a small percentage of T cells, we were able to demonstrate the specific contribution of LC by using CD1a isolation and CD3 depletion.
Coinfections with pathogens activate LCs through TLR triggering. Therefore, we investigated whether different bacterial TLR agonists influence HIV-1 transmission in the ex vivo model. We demonstrated that TLR2, TLR4, and TLR5 agonists increase HIV-1 transmission in tissues derived from some donors, whereas no increase was observed in other donors. Notably, the TLR4 agonist LPS did induce HIV-1 transmission by LCs in some donors, even though LCs do not express TLR4 (L. de Witte and M.A.W.P. de Jong, unpublished observations) (20, 21). These data suggest that the observed increase for at least the TLR4 agonist is due to an indirect activation of LCs, such as production of proinflammatory cytokines by keratinocytes, since keratinocytes and epithelial cells do express TLR4 (20). Indeed, we observed the production of TNF-α after LPS stimulation of skin biopsies. This might be variable throughout donors due to different levels of TLR expression and activity, resulting in the variation in HIV-1 susceptibility (16). The TLR5 agonist flagellin enhanced HIV-1 transmission in our ex vivo model in half of the donors. However, the effect of flagellin was indirect, since the increased transmission was abrogated after washing the emigrated LCs and flagellin did not increase HIV-1 capture or replication in LCs as determined by RT-PCR (L. de Witte and M.A.W.P. de Jong, unpublished observations). Moreover, flagellin itself did not increase susceptibility of CCR5+ Jurkat T cells (L. de Witte and M.A.W.P. de Jong, unpublished observations), suggesting that flagellin induces the production of a soluble factor by surrounding cells that enhances infection of T cells. Furthermore, TNF-α neutralization by antibodies in the ex vivo experiment did not prevent the increase of transmission by flagellin (Supplemental Figure 8), suggesting that TNF-α is not involved in the enhanced transmission by flagellin.
In contrast to the other TLR agonists, Pam3CSK4 enhanced transmission in all donors tested. Pam3CSK4 is a synthetic tripalmitoylated lipopeptide that mimics the acetylated amino terminus of bacterial lipoproteins (42, 43). Recognition of Pam3CSK4 is mediated by a dimer of TLR1 and TLR2 (44). LCs and keratinocytes both express TLR1 and TLR2 (20, 21, 45). Neutralizing antibodies against TNF-α did not abrogate the Pam3CSK4 induction of HIV-1 transmission (Supplemental Figure 8), demonstrating that Pam3CSK4 increased transmission independent of TNF-α. Using a single-cycle replication-defective HIV-1, we demonstrate that the increase in HIV-1 transmission by Pam3CSK4-stimulated LCs is independent of HIV-1 infection of LCs, demonstrating that the increase is at least partly due to trans-infection of T cells; HIV-1 capture by LCs is increased and the surface-bound viruses are efficiently transmitted to T cells. This is supported by our finding that HIV-1 capture by LCs is strongly increased after Pam3CSK4 stimulation. Single particles and clusters of HIV-1 are observed intracellularly and on the cell membrane, some colocalizing with HLA class I molecules, which might reflect exosomes. Although HIV-1 was observed in intracellular vesicles, cell-bound HIV-1 was primarily transmitted after Pam3CSK4 stimulation. Pam3CSK4 might upregulate the expression of receptors that increase HIV-1 capture, such as heparan sulfate proteoglycans (46). However, heparinase treatment did not abrogate HIV-1 transmission after Pam3CSK4 treatment (L. de Witte and M.A.W.P. de Jong, unpublished observations), indicating that heparan sulfates are not involved. Possibly, Pam3CSK4 enhances LC activation, which results in increased HIV-1 binding. Notably, enhanced HIV-1 capture by Pam3CSK4 is observed within 30 minutes (L. de Witte and M.A.W.P. de Jong, unpublished observations) and Pam3CSK4-stimulated LCs rapidly change phenotypically and form clusters. A recent report demonstrated that Pam3CSK4 stimulation in T cells results in the activation of NF-_B and upregulation of the expression of CCR5. After 24 hours of stimulation, these activated T cells are more susceptible to both X4- and R5-tropic HIV-1 (47). However, we observed increased trans-infection, which is independent of fusion of HIV-1 with LCs, strongly suggesting that increased HIV-1 capture but not infection is responsible for transmission after Pam3CSK4 stimulation.
TLR agonists as well as whole pathogens can increase HIV-1 transmission indirectly by changing the cytokine environment. Indeed, TLR agonists, Neisseria gonorrhea and the different forms of Candida albicans induced the production of proinflammatory cytokine TNF-α in vaginal and skin tissues ex vivo. Therefore, we investigated what the effect of proinflammatory cytokine TNF-α was on HIV-1 transmission. Our data demonstrate that TNF-α strongly upregulated HIV-1 transmission by LCs ex vivo, suggesting that LC activation by TNF-α leads to increased susceptibility to HIV-1 under coinfection circumstances. Further study of the molecular mechanism demonstrates that TNF-α increased the level of HIV-1 replication in infected LCs, which results in enhanced HIV-1 transmission of de novo produced viruses to T cells. This is further supported by the need for HIV-1 replication in LCs, since TNF-α- in contrast to Pam3CSK4-treated LCs do not transmit replication-defective HIV-1 to T cells ex vivo. Moreover, TNF-α did not increase HIV-1 capture by LCs but enhanced initiation of HIV-1 transcription as determined by the increase in the multiply spliced transcripts of the early HIV-1 genes tat and rev. These data are supported by previous studies demonstrating that TNF-α enhances replication of HIV-1 in T cells and macrophages (27). These data strongly suggest that coinfections abrogate the protective barrier function of LCs and allow LC infection and subsequent HIV-1 transmission. Inflammation not only affects LC function but also epithelial and sometimes subepithelial tissues, resulting in the production of inflammatory factors, influx of immune cells, ulceration, and bleeding. Different mechanisms have been proposed to explain the increased risk of HIV-1 acquisition during coinfection such as genital ulceration (6). However, we here demonstrate that the role of LCs should be taken into account.
We have previously shown that Langerin protects LCs from HIV-1 infection and thereby prevents HIV-1 transmission (16). Here we have used high viral concentrations in the ex vivo transmission model, which were previously shown to saturate the Langerin function (16). Titration of HIV-1 in the transmission assays suggests that Pam3CSK4 but not TNF-α activation overcomes the protective function of Langerin. Pam3CSK4 induced viral transmission by LCs even at low concentrations when Langerin was not saturated. In contrast, TNF-α only increased HIV-1 transmission at concentrations that already allowed HIV-1 transmission under normal conditions. Since TNF-α enhances HIV-1 transcription, low levels of infection are a prerequisite for TNF-α to enhance HIV-1 transmission and therefore dependent on the Langerin function. In contrast, Pam3CSK4 enhances capture of HIV-1 for transmission to T cells. Therefore, at low viral loads, there could be competition between (unknown) surface attachment receptors and Langerin for HIV-1. Our data suggest that LC activation by Pam3CSK4 is strong enough to negate the protection by Langerin. From the observed mechanisms, we speculate that the presence of TNF-α is extremely important when Langerin function has been compromised and initial infection has taken place. In contrast, TLR1/TLR2 activation by pathogens during coinfections will overcome the Langerin barrier and induce HIV-1 transmission by LCs through increased HIV-1 uptake and transmission to T cells, thereby strongly enhancing the susceptibility of the host to HIV-1 infection. In our model, no differences in surface Langerin expression were observed after addition of TNF-α or Pam3CSK4. However, we cannot exclude that the degradation function or clustering of Langerin is hampered in the presence of these compounds. Further studies will be necessary to investigate the effects of LC activation on Langerin function.
Since the development of an effective vaccine or a curative treatment for HIV-1 is not progressing rapidly, the WHO stated that focus should be on the development of an agent that prevents HIV-1 transmission and that can be applied topically, such as a genital cream (1). Our results indicate that coinfection results in a changed LC phenotype that can result in transmission, mediated through different mechanisms. To prevent LC infection and subsequent transmission, it is attractive to try to block the entry receptors of HIV-1 on the LCs, such as CD4 and CCR5 (48). However, TLR activation by coinfecting pathogens might increase HIV-1 transmission by yet unknown mechanisms, such as the increased capture of HIV-1 and subsequent trans-infection observed with Pam3CSK4 treatment. Since these mechanisms are potential targets for therapies these need to be further unravelled. Since TNF-α enhances LC infection and subsequent transmission, the cytokine itself or its receptor represent targets to prevent HIV-1 transmission. Moreover, these results emphasize the argument that prevention and fast intervention to treat coinfections and especially chronic coinfections should be taken as a serious point in the prevention of HIV-1 transmission.
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