Antimicrobial β-defensins, expressed at high levels in mucosae, are an important component of innate immunity in epithelia. Our results show that hBD-2 and 3 possess dose-dependent HIV-suppressive activity, while hBD1 is significantly less active. The antiviral activity was reproducibly observed with three isolates of HIV, two of which utilize CXCR4 (IIIB and NL4.3), while the third is CCR5 tropic (BaL), thus suggesting a broad-spectrum antiviral activity (Fig. and ). Interestingly, we noticed that, at least in the case of the isolates tested, hBD2 was more efficient in inhibiting replication of the X4 isolates (Fig. and ). These results are in accordance with the result of a recently published study (39
). We observed some striking differences between our results and those of Quiñones-Mateu et al. (39
). First, all of our data were gathered under high-salt, serum-rich conditions. Under high-salt conditions, the previous study detected no inhibition of R5 isolates, and even under low-salt conditions the inhibition of R5 isolates was marginal (39
). In contrast, in our study inhibition of R5 isolates was very pronounced (Fig. ), and one would expect to see even higher inhibition in serum-free conditions, as serum can sequester defensins (38
). In addition, the inhibitory effect of hBD3 was accompanied by some level of cellular toxicity, especially at higher doses (100 μg/ml), so that we could not discriminate how this toxicity would affect viral replication, and we chose to compare hBD1 to hBD2 for further studies. It is conceivable that the different source of peptides (baculovirus-or Escherichia coli
-expressed as opposed to synthetic) or the target cells used (the cell line GHOST, expressing HIV coreceptor, as opposed to PBMC) could account for the discrepancies that we observed. Our attempt to dissect the mechanism of action of these peptides yielded some unexpected results.
Defensins are known to disrupt membrane structure (16
), and θ-defensins tightly bind to the sugar moieties on gp120 and CD4 (12
), so that we envisioned a mechanism of inhibition directed to both virus and cells. Our main hypothesis when we started our studies was that these effects would concur in inhibiting membrane fusion events mediated by HIV envelope. Consistently with a blockade of HIV at an early stage, we observed that concentrations comparable to those used in the infectivity assays could inhibit the accumulation of early products (4 h after infection) of reverse transcription, using both X4 and R5 isolates of HIV as detected by a quantitative real-time PCR assay (Fig. ). To our surprise, however, we could not detect any reproducible inhibition of env
-mediated fusion, despite the use of different experimental approaches to study the process and of both cell lines and primary cells. We evaluated the expression of coreceptors CD4, CXCR4, and CCR5 on the cell membranes by flow cytometry and did not observe any modulation following treatment with hBD1 and hBD2 in a range of concentrations between 20 and 0.8 μg/ml in both resting and PHA-activated cells (Fig. ). This finding is at odds with the report by Quiñones-Mateu et al., who described a downregulation of CXCR4 upon hBD2 treatment (39
). The only significant difference in their studies is the use of recombinant defensins, as opposed to our use of synthetic defensins. It is possible that the two molecules of different origin might have a different activity, and a comparative study will elucidate this discrepancy. Of note, lipopolysaccharide has been shown to downregulate CXCR4, although this is a potential contaminant of proteins expressed in bacteria and should therefore not be present in baculovirus-expressed proteins (52
). We also studied release of HIV inhibitory chemokines, which have been described to mediate the anti-HIV activity of α-defensins in macrophages (19
), and found that only hBD1 treatment results in some increase in RANTES release in PBMC, while none of the other chemokines showed detectable increases as measured by ELISA.
We could detect some decrease in HIV infectivity, but this decrease was not specific for hBD2, as also hBD1 treatment lowered HIV infectivity (Fig. ). Therefore, while we think that direct inactivation of HIV might contribute to overall suppression, this is probably not the only mechanism responsible for it. Confirmation for this possibility comes from time course experiments where we treated cells from 10 to 120 min after infection; control infections were left untreated, while control infections for inhibition by defensins were treated prior to and at the time of infection, as in our pilot experiments. In addition, we treated cells with agents known to block fusion intermediates. As expected, the efficacy of these agents in blocking HIV infection decreased dramatically after 60 to 90 min after infection had been started (Fig. ). In contrast, the efficacy of hBD2 remained ~80% even when treatment was started 120
min after infection (Fig. ). The significantly lower efficacy of treatment with the same concentration of hBD1 (~20%) at all time points indicated that the inhibition is not a specific phenomenon. The finding that hBD2 inhibits HIV replication postinfection is of interest. Importantly, this result was obtained in a 16-h infection so that the observed suppression was not due to inhibition of spread. β-Defensins are known to signal through several receptors. For example, human α- and β-defensins selectively chemoattract different subsets of T lymphocytes and immature dendritic cells, thus playing important roles as immune modulators in adaptive immunity as well (10
). For hBDs, chemotaxis of immature dendritic cells and memory T cells results from their direct binding and activation of the chemokine receptor CCR6, whose only known chemokine-ligand is MIP-3α (59
). In addition, β-defensins induce intracellular signaling by interacting with chemokine and Toll-like receptors (5
), so that we cannot rule out that additional, late events in HIV replication might also be affected by β-defensins. These results are consistent with a model of inhibition by hBD2 due to either a receptor-triggered event or a change in membrane dynamics that, without affecting fusion events, results in inhibition of some signaling event. For example, disrupting membrane rafts can block signaling events. This model is compatible with observations made by Chang et al. (9
), who studied the HIV-suppressive activity of α-defensins 1 to 3, and it is conceivable that a common or parallel mechanism of HIV inhibition exists between α-defensins and hBD2.
The range of concentration of hBD2 tested, between 100 and 0.8 μg/ml (i.e., in the 0.2 to 25 millimolar range), might seem high in comparison to the antiviral activity of other known soluble inhibitors, such as CCR5 ligands, which are active in the 10 to 100 μM range (11
). However, it must be noted that this is the same concentration range for antibacterial activity. Further, expression of β-defensins at levels comparable to the ones used in our study has been described in the oral mucosa (49
). Since transmission of HIV seems to occur less readily through the oral mucosa, we hypothesize that β-defensins might constitute a component of the innate immunity to this virus. In addition, it is possible that the immunodeficiency triggered by HIV infection decreases expression of this defensive mechanism, thus increasing occurrence of oral complications often observed in the course of HIV disease. In this respect, using immunohistochemical analyses, we observed that hBD2 expression, which in healthy individuals not infected with HIV is expressed at high levels in the epithelium of the oral mucosa and forms a thick barrier, is dramatically decreased in HIV-positive subjects, potentially leaving them more likely to contract opportunistic infections or other oral complications (Fig. ).
The elucidation of the HIV-inhibitory activity of β-defensins and of their pattern of expression in the oral mucosa of HIV-negative and HIV-positive subjects has several important consequences. First, it is now imperative to investigate the expression of these proteins in mucosae from oral, rectal, and vaginal tissue, since it is possible that differential levels of expression of β-defensins might contribute to explain the documented lower rate of infection through oral mucosa. Further, it is conceivable that the oral transmission observed in infant macaques could be due to a less efficient expression of β-defensins in the oral mucosa in infants compared to adults, so that an investigation of the relative levels of expression in these two populations is warranted. Interestingly, a recent study has found a correlation between a polymorphism in the hBD1 gene and risk of HIV-1 infection in a population of 97 children (7
). Finally, the finding that molecules that are constitutively expressed in the mucosa can inhibit HIV without being associated with inflammation has important consequences for both preventive and therapeutic approaches. In prevention, β-defensins or derivatives or small molecules modeled on them could be included in topical microbicide preparations; the same molecules could be also be evaluated in the growing arsenal of weapons to be used to treat HIV infection.