This study shows that the negatively charged polyanionic microbicidal compounds DxS and PSS significantly modify the outcome of TLR stimulation in cervical and vaginal epithelial cells, including cytokine/chemokine responses and NF-κB activation.
TLR activation is critical for epithelial immune responses and the elimination of pathogen invasion. TLRs trigger innate epithelial defense mechanisms, including the production of antimicrobial peptides (64
) and antiviral cytokines, e.g., IFN-β, which is produced by virus-infected cells and increases bystander cell resistance to viral infection (9
). In addition, TLR responses are needed for activation of adaptive immune responses via production of IL-1, IL-6, IL-8, tumor necrosis factor alpha, and IL-12 and upregulation of immune cell costimulatory molecules, e.g., CD80, CD86, and CD40 (43
). The production of IL-8 by epithelial cells in response to TLR stimulation serves to attract and activate innate immune cells, such as neutrophils, to the site of infection and inflammation, which is necessary for bacterial infection clearance (31
). Thus, the inhibition of cytokine/chemokine production and/or secretion could lead to impaired protective responses and increased risk of pathogen invasion.
The polyanionic microbicide compounds characterized in this study inhibited the production and release of IL-8 in response to the TLR1/2 agonist Pam3CSK4 while having no or only a weak impact on the TLR2/6 agonist MALP-2. These effects occurred in spite of the significantly enhanced NF-κB activation in the presence of both compounds, suggesting a pathway-specific modulation downstream from receptor-ligand interactions.
DxS and PSS radically suppressed multiple steps of the TLR3 pathway, including ligand uptake, NF-κB activation, and cytokine mRNA and protein production. In contrast to TLR1/2 and TLR2/6, TLR3 was inhibited both upstream and downstream from NF-κB activation. Because of the intracellular localization of TLR3, poly(I:C) must be internalized in order to initiate TLR signaling. The polyanionic compounds prevented poly(I:C) internalization and binding to TLR3, since the NF-κB inhibitory effect was seen only when the compound and the ligand were added simultaneously to the epithelial surface and not in experiments where epithelial cells were incubated with poly(I:C) prior to compound exposure. However, cytokine production was still reduced in the latter experiments, suggesting that the immunosuppressive mechanism also included events downstream from poly(I:C) uptake, TLR binding, and NF-κB activation. An additional proof for this hypothesis is that DxS inhibited the IL-8 and IL-6 responses to poly(I:C) in the VEC-100 model, where poly(I:C) was added to the basal surface and DxS was applied to the apical surface of the tissue. A recent study identified scavenger receptor class A (SR-A) as a novel epithelial cell surface receptor responsible for the internalization of poly(I:C) (40
). Furthermore, the same study showed that polyanionic competitors of SR-A, such as DxS, inhibit poly(I:C)-SR-A binding, uptake, and proinflammatory response. Our data show that these upstream effects of the polyanionic compounds are different for poly(I:C) and for Pam3
and MALP-2. While the presence of polyanions inhibited the poly(I:C)-triggered NF-κB response, it potentiated responses to simultaneously added Pam3
and MALP-2, which bind cell surface TLR1/2 and TLR2/6, respectively. This enhancement of NF-κB activation might be due to potentiated TLR-ligand binding or might be a result of autocrine stimulation by cell membrane-retained cytokines.
It is of critical importance to understand the mechanisms underlying modified TLR-mediated responses in the presence of microbicidal agents, since they may be responsible for significant unwanted consequences during microbicide use for prevention of HIV transmission. The fact that both polyanionic compounds characterized in this study, DxS and PSS, had similar effects on TLR-mediated IL-8 secretion, while unsulfated Dx had no effect, suggests that their effects on TLR signaling are possibly related to their negative charge, which also underlies their main mechanism of anti-HIV-1 action. Both DxS and PSS exert anti-HIV-1 activity through binding to the positively charged sites in the V3 loop of the viral envelope glycoprotein gp120 and blocking its interaction with CD4 receptors of HIV-1 target cells (44
). Unsulfated Dx, which does not display antiviral activity, did not have any significant impact on TLR-mediated cytokine production and secretion.
The HIV-1 neutralization mechanism of DxS, PSS, and other sulfated polyanions also includes blocking the primary binding of HIV-1 gp120 to cell surface heparan sulfate (45
). By competing with heparan sulfate on a larger scale, these compounds may interfere with a number of important immunoregulatory mechanisms. Sulfated disaccharides derived from heparin/heparan sulfate have been reported to downregulate the spontaneous and tumor necrosis factor alpha-stimulated secretion of IL-8 and IL-1β by intestinal epithelial cells (14
). Their mechanism of action, however, has not been established fully (11
An increasing number of cytokines have been found to bind not only to their high-affinity receptors but also to cell surface glycosaminoglycans from the heparin/heparan sulfate family (47
). The binding of a cytokine to heparan sulfate may have a number of functions, either facilitating or inhibiting the cytokine's biological activity. DxS and PSS may act as synthetic analogs of heparan sulfate and other sulfated glycosaminoglycans, which regulate the function of chemokines, e.g., IL-8 (59
). The sequestration of proinflammatory cytokines at the cell surface has been shown to induce the synthesis and secretion of heparanase, an enzyme that cleaves heparan sulfate in epithelial and endothelial cells and has proinflammatory activity (13
). Thus, the polyanion sequestration of cytokines may have anti-inflammatory but also proinflammatory consequences and deserves further investigation.
As a result of the interaction of polyanionic microbicides with the female genital tract epithelium, reduced levels of TLR-stimulated cytokines and chemokines may attenuate potential inflammatory reactions. Although the process of mucosal immune regulation is complex and requires further study, given the known HIV activation by inflammatory conditions and cytokines, the compound-induced reduction of the proinflammatory cytokine response in healthy individuals in the absence of bacterial infection may be regarded as beneficial. However, at the same time, the decreased production of cytokines would impair the protective and regulatory paracrine function of the cervicovaginal mucosa, rendering it more susceptible to bacterial and viral infections. Decreased levels of vaginal cytokines and certain microflora abnormalities have been reported in clinical studies upon application of polyanionic microbicides (10
). Our study offers a biological explanation for these observations and presents a model system for future assessment of other microbicides targeting host receptor interactions. The clinical significance of this in vitro model remains to be confirmed by future outcomes of microbicide trials.
Since TLR activation enhances HIV-1 replication (6
), it might be argued that inhibition of TLR by microbicidal compounds could be beneficial. However, women at higher risk of HIV-1 are also exposed to other sexually transmitted viral and bacterial pathogens, and inefficient stimulation of TLRs, which is the first innate immune response to infection, might increase the risk of microbial invasion, which in turn would lead to increased risk for HIV-1 infection. Thus, impaired TLR responses, especially those lasting after the microbicides are washed out and are no longer present in the vagina at a protective dose, may ultimately increase the risk of HIV-1 transmission. A recent study showed that TLR tolerance induced by TLR ligands enhances HIV-1 gene expression (5
). Therefore, it seems safer for microbicide products to not interfere with epithelial TLR responses.
Our in vitro findings suggest that polyanionic microbicidal compounds may interfere with TLR responses and affect the ability of the cervicovaginal epithelium to mount normal responses against microbial pathogens. Given the diverse effects of TLR signaling on HIV-1 transmission, further studies are required to determine the clinical relevance of these interactions.