Chlamydia trachomatis Enhances HIV Infection of Non-Activated PBMCs

Abstract

Sexual contact is the most common route of HIV transmission, and the concurrent presence of sexually transmitted infections (STIs) such as Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (gonococcus, GC) is known to increase the HIV risk. Antibiotic treatment decreases the incidence of STIs but not HIV. CT and GC activate Toll-like receptors (TLRs) 2 and 4, which act as sensors of microbial infection are critical for initiating immune responses to control infection. We have previously shown that GC enhances HIV infection of primary resting CD4+ T cells through activation of TLR2 but not TLR4. In this study, we determined the effect of live and fixed CT and different species of lactobacilli including L. jensenii and L. reuteri on HIV infection of freshly isolated PBMCs. We found that pretreatment of freshly isolated PBMCs with fresh or fixed CT, but not lactobacilli, promoted HIV infection of freshly isolated CD4+ T cells. Together with our previous reports, we concluded that STIs such as CT and GC but not commensal bacteria like lactobacilli enhanced HIV infection, possibly through immune activation. Importantly, the enhancement effect of fixed CT on HIV infection may explain the failure of antibiotic treatments to reduce the HIV incidence. Combined strategies to inhibit STI growth and STI-mediated mucosal immune activation should be considered for HIV prevention in the settings of STIs.

Introduction

Sexual transmission is the main route of HIV infection, and sexually transmitted infections (STIs) including Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (gonococcus, GC) increase the likelihood of HIV transmission [1–4]. Antimicrobial therapies for bacteria or herpes simplex virus 2 reduce the incidence of STIs but not HIV [5,6]. The contribution of STIs to the increase in HIV transmission is likely to be multifaceted (reviewed in [7,8], understanding how STIs enhance HIV infection is vital to the development of new strategies to reduce the spread of HIV. CT and GC are known to activate Toll-like receptors (TLRs) 2 and 4 [9–11] that detect microbial infection and are critical for the initiation of innate inflammatory and adaptive immune responses [12–14]. Induction of mucosal innate immune response by non-ulcerative STIs such as CT or GC may lead to enhanced HIV transmission. Indeed, we have previously shown that GC promotes HIV infection of primary resting CD4+ T cells through activation of TLR2 but not TLR4 [15]. In this study, we compared the effect of live and fixed CT on HIV infection of freshly isolated PBMCs. Lactobacillus jensenii and L. reuteri, commensal bacteria in the vagina and gut, respectively, were included as a comparison. Our data showed that live and fixed CT but not lactobacilli promoted HIV infection of freshly isolated PBMCs, suggesting that strategies targeting STI-mediated immune activation in addition to antimicrobial therapies are needed for HIV prevention.

Materials and Methods

Reagents

Recombinant human interleukin 2 (IL-2) was purchased from R&D systems (Minneapolis, MN). Peptidoglycan (PGN), Histopaque®−1077, RPMI-1640 medium and CsA were from Sigma-Aldrich (St Louis, MO).

Pre-treatment of PBMCs with bacteria, and HIV-1 infection assay

Peripheral blood mononuclear cells (PBMCs) from normal healthy blood donors were isolated over a Histopaque gradient. Cells were cultured in RPMI-1640 media with 10% FBS in the presence of IL-2 (50 IU/ml). Replication-defective pseudotyped HIV-1_VSV-G luciferase reporter viruses were produced as described previously [16,17]. Live or fixed extracellular, infectious elementary bodies of Chlamydia trachomatis serovar D (D/ UW3/Cx) were kindly provided by Dr. Alison J. Quayle at Louisiana State University Health Sciences Center, New Orleans, Louisiana. Lactobacilli were cultured in MRS broth (Difco) at 37^oC in 5% CO₂ as described previously [18]. Bacterial cell density was adjusted to an OD₆₀₀ nm equal to 0.1, corresponding to 10⁸ colony forming units/ml bacteria. Fixed bacteria were prepared by using 1% paraformaldehyde in PBS as described previously [15].

Freshly isolated PBMCs (1 × 10⁶ cells per sample) were treated with bacteria in the presence of IL-2 for 2 days. As a control, PBMCs were treated with PGN (20 μg/ml) as described previously [15]. Cells were washed and then infected with pseudotyped CCR5-using HIV-1_JR-FL luciferase reporter virus at 37°C for 2h. After washing off unbound viruses, infected cells were cultured in the presence of IL-2 at 37°C for 3 days. Cells were then lysed with passive cell lysis buffer (Promega, Madison WI) and luciferase activity (in relative light units [RLUs]) was measured on a Glomax 20/20 luminometer (Promega, Madison WI).

Results and Discussions

To determine whether CT increased the susceptibility of PBMCs to HIV, we treated freshly isolated PBMCs with live and fixed CT at MOI (multiplicity of infection) of 10 in the presence of IL-2 for 2 days before HIV infection. Live and fixed L. jensenii and L. reuteri at MOI of 1 and 10 were included as a comparison. PGNs were included as a positive control as we have previously shown that pretreatment of primary CD4+ T cells with PGNs enhances HIV infection [15]. As shown in figure 1, there was a low level of HIV infection of non-activated PBMCs. In agreement with our previous finding, the degree of HIV infection of PGN-treated PBMCs was higher than that of untreated cells (10-fold). Live CT slightly, but not significantly, increased HIV infection. Fixed CT significantly increased HIV infection by 6-fold compared to untreated PBMCs. L. jensenii and L. reuteri did not promote HIV infection regardless whether live or fixed bacteria were used. In fact, lactobacilli suppressed the low level of baseline HIV infection.

Figure 1:

Chlamydia trachomatis but not Lactobacillus species enhance HIV infection of non-activated PBMCs. Freshly isolated PBMCs were pretreated with live or fixed CT, L. jensenii (L.j) or L. reuteri (L.r) at MOI 1 or 10 in the presence of IL-2 for 2 days. Cells were treated with PGN (20 μg/ml) as a positive control. PBMCs without bacterial pretreatment or without HIV infection were included. PBMCs were then infected with pseudotyped HIV-1JR-FL luciferase reporter virus and HIV infection was determined by measuring luciferase activity at day 3 after infection. The difference between samples with treatment of PGN or fixed CT vs samples without treatment is significant (*p < 0.05).

We have previously shown that GC and TLR2 signal activate primary CD4+ T cells [15,19]. Since primary CD4+ T cells did not respond to TLR4, the enhanced HIV infection by fixed CT is likely mediated through TLR2. The differential effect of live and fixed CT on HIV infection may be due to CT-induced immune responses from other cell types such as monocytes that may have an indirect effect on CD4+ T cells. Studies using primary CD4+ T cells alone or stimulation of specific immune cells with CT followed by co-culture systems will provide insights into the effect of CT on HIV infection in the mixed cell-type culture.

The ability of lactobacilli to induce TLR activation has been in consistent. Some reports indicated that lactobacilli are involved in TLR2, 4, 5, and 9 [20,21] but others show little activation or suppression of TLR4-medated response. For example, in Caco-2 cells, Lactobacillus rhamnosus GG down-regulated the enterotoxigenic Escherichia coli-mediated induction of IL-1β and TNF-α [22]. Further investigation is needed to confirm the suppressive effect of lactobacilli on HIV infection in both non-activated and activated PBMCs and to determine the underlying mechanism.

CT increases HIV transmission through several mechanisms. CD4+ T cells are recruited in the endocervix in women with CT [23]. CT infection of endocervical cells facilitates HIV crossing of mucosal barrier and subsequent infection [24,25]. CT also modulates mucosal immune milieu including antimicrobial peptides including defensins 5 and 6, which promote HIV infectivity [26]. Our result showed that CT promoted HIV infection of PBMCs. Interestingly, similar to our previous finding indicating that GC products increased HIV infection via TLR2 activation [15], fixed CT promoted HIV infection of freshly isolated PBMCs. Our results may explain why antibiotics reduce the incidence of bacterial STIs but not HIV [6] as dead bacteria can activate TLR signaling.

Conclusion

In conclusion, we found that live and fixed CT but not lactobacilli enhanced HIV infection. Together with our previous findings using GC and GC products that activate TLR2 to promote HIV infection, blocking STI-mediated immune activation in addition to the antibiotic treatment may offer a better strategy to prevent HIV spread in women with asymptomatic STIs.