Trappin-2/Elafin: a novel innate anti-human immunodeficiency virus-1 molecule of the human female reproductive tract - PubMed (original) (raw)

Trappin-2/Elafin: a novel innate anti-human immunodeficiency virus-1 molecule of the human female reproductive tract

Mimi Ghosh et al. Immunology. 2010 Feb.

Abstract

Trappin-2/Elafin is a serine protease inhibitor that plays a major role as an anti-inflammatory mediator at mucosal surfaces. In addition, Trappin-2/Elafin has antibacterial activity against Gram-positive and Gram-negative bacterial and fungal pathogens. In this study we examined the production of Trappin-2/Elafin by epithelial cells from the human upper and lower female reproductive tract as well as its activity as an anti-human immunodeficiency virus (HIV)-1 molecule. We found that primary uterine, Fallopian tube, cervical and ectocervical epithelial cells produce Trappin-2/Elafin constitutively and that production of Trappin-2/Elafin is enhanced following stimulation with Poly(I:C), especially by the uterine cells. Given the presence of Trappin-2/Elafin in the reproductive tract, we tested the ability of recombinant Trappin-2/Elafin to inhibit HIV-1, an important sexually transmitted pathogen. We found that recombinant Trappin-2/Elafin was able to inhibit both T-cell-tropic X4/IIIB and macrophage-tropic R5/BaL HIV-1 in a dose-dependent manner. The inhibitory activity was observed when virus was incubated with Trappin-2/Elafin but not when Trappin-2/Elafin was added to cells either before infection or after infection. This suggests that the mechanism of inhibition is likely to be a direct interaction between HIV-1 and Trappin-2/Elafin. Additionally, we measured the levels of secreted Trappin-2/Elafin in cervico-vaginal lavages (CVL) from both HIV-positive and HIV-negative women and found that average levels of secreted Trappin-2/Elafin were higher in the CVL from HIV-negative women, although the values did not reach statistical significance. We also found that women at the secretory phase of the menstrual cycle produced more Trappin-2/Elafin in CVL relative to women at the proliferative phase of the menstrual cycle. Our data suggest that Trappin-2/Elafin might be an important endogenous microbicide of the female reproductive tract that is protective against HIV-1.

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Figures

Figure 1

Figure 1

Constitutive production of Trappin-2/Elafin by epithelial cells of the female reproductive tract. (a) Primary uterine (UT), Fallopian tube (FT), cervical (Cx) and ectocervical (Ecx) epithelial cells were isolated from four patients and cultured until confluence and high transepithelial resistance (TER) was reached (with the exception of Ecx cells, which do not polarize). RNA was extracted from the cells and real-time reverse transcription–polymerase chain reaction (RT-PCR) was used to determine the relative expression levels of Trappin-2/Elafin. After normalization to endogenous control β-actin, each patient sample was further calibrated to Ecx cells, which typically produced low levels of Trappin-2/Elafin. The data are shown relative to Ecx, which was set at 1. Generally, FT cells produced significantly higher levels of Trappin-2/Elafin mRNA compared with Cx, Ecx and UT cells. All values twofold higher or lower were considered to be significant. (b) After 24 hr, accumulation of constitutive apical and basolateral conditioned media was collected from female reproductive tract (FRT) epithelial cells from patients and assayed for Trappin-2/Elafin protein secretion by enzyme-linked immunosorbent assay (ELISA). An average of three to five patients per each tissue is shown. Significantly higher levels of constitutive Trappin-2/Elafin secretion were observed in the fallopian tube epithelial cells (FTEC) compared with UT, Cx and Ecx cells. The _P_-values were calculated using analysis of variance (

anova)

; ***significantly greater than control, P < 0·001.

Figure 2

Figure 2

Induction of Trappin-2/Elafin messenger RNA (mRNA) and protein by uterine (UT) epithelial cells upon treatment with Toll-like receptor 3 (TLR3) agonist Poly(I:C). (a) Primary UT epithelial cells from six different patients were treated with Poly(I:C) for 24 hr after which the cells were harvested and RNA extracted. Real-time reverse transcription–polymerase chain reaction (RT-PCR) was used to determine the relative expression levels of Trappin-2/Elafin. After normalization to endogenous control β-actin, each patient sample was further calibrated to its own untreated control. In all six UT samples, Trappin-2/Elafin mRNA levels were enhanced by at least twofold. (b) Primary UT epithelial cells were treated with Poly(I:C) for 3, 6 and 24 hr before the cells were harvested and RNA was extracted. Real-time RT-PCR was used to determine the relative expression levels of Trappin-2/Elafin. Data were normalized to endogenous control β-actin and further calibrated to its own untreated control. Upon Poly(I:C) treatment, Trappin-2/Elafin mRNA was found not to be upregulated at 3 hr, but was upregulated at 6 hr and the upregulation was maintained at 24 hr. Data are shown from one out of three representative patients. (c) UT epithelial cells were treated with Poly(I:C) for 24 hr. Apical and basolateral conditioned media (CM) were collected and assayed for Trappin-2/Elafin protein secretion by enzyme-linked immunosorbent assay (ELISA). Secretion of Trappin-2/Elafin upon Poly(I:C) treatment was significantly enhanced when compared with untreated controls. Data from one representative patient out of four is shown. The _P_-values were calculated using a two-tailed paired _t_-test. *Significantly greater than control, P < 0·05.

Figure 3

Figure 3

Direct anti-human immunodeficiency virus-1 (HIV-1) activity of recombinant Trappin-2/Elafin. Human recombinant Trappin-2/Elafin at 0·01, 0·1, 1 and 10 ng/ml was incubated with HIV-1 IIIB and BaL at a multiplicity of infection (MOI) of 1 for 1 hr at 37° before addition onto TZM-bl cells. Substrate β-Glo was added 48 hr postinfection and the level of viral infection was quantified using a luminometer. Data were expressed as ‘per cent of control’ with the ‘virus-only’ samples set at 100%. Trappin-2/Elafin was found to significantly inhibit both HIV-1 IIIB and BaL at all doses tested. The _P_-values were calculated using a two-tailed paired _t_-test. Significantly greater than control: ***, P < 0·001.

Figure 4

Figure 4

Anti-human immunodeficiency virus-1 (HIV-1) activity of Trappin-2/Elafin did not occur through interaction with TZM-bl cells or through postinfection inhibition mechanisms. (a) Human recombinant Trappin-2/Elafin at 0·1 and 1 ng/ml was added to TZM-bl cells for 1 hr at 37° followed by washing the cells and adding HIV-1 IIIB and BaL at a multiplicity of infection (MOI) of 1. Substrate β-Glo was added 48 hr postinfection and the level of viral infection was quantified using a luminometer. Data were expressed as ‘per cent of control’ with the ‘virus-only’ samples set at 100%. Trappin-2/Elafin did not significantly inhibit either IIIB or BaL HIV-1 when added to the cells before infection. (b) TZM-bl cells were infected with HIV-1 IIIB and BaL at MOI of 1. At 6 and 24 hr postinfection, cells were washed and 1 ng/ml of recombinant Trappin-2/Elafin was added. Assays were developed by addition of substrate β-Glo and viral infection was quantified using a luminometer. Data were expressed as ‘per cent of control’ with the ‘virus-only’ samples set at 100%. With the exception of a slight inhibition observed 24 hr after infection with HIV-1 IIIB, no anti-HIV-1 activity was observed under any of the conditions.

Figure 5

Figure 5

Trappin-2/Elafin protein levels were higher in cervico-vaginal lavages (CVL) obtained from human immunodeficiency virus (HIV)-negative women when compared with HIV-positive women and regulated by the menstrual cycle. (a) Supernatants from CVL obtained from 32 HIV-positive women and 15 HIV-negative women were tested for Trappin-2/Elafin protein by enzyme-linked immunosorbent assay (ELISA). The average for the HIV-negative women was found to be higher than the average for the HIV-positive women. (b) When the patients were stratified by race, the same trend was observed; in each case average levels of Trappin-2/Elafin for the HIV-negative women were higher than those for the HIV-positive women. (c) When HIV-positive patients were stratified according to menstrual status, those in the secretory phase of the cycle produced significantly more Trappin-2/Elafin than those in the proliferative phase of the cycle. The data in this case were normalized to total protein content in each sample and are expressed as pg of Trappin-2/Elafin per μg of protein.

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References

    1. Padian NS, van der Straten A, Ramjee G, et al. Diaphragm and lubricant gel for prevention of HIV acquisition in southern African women: a randomised controlled trial. Lancet. 2007;370:251–61. - PMC - PubMed
    1. Cohen J. AIDS research. Promising AIDS vaccine’s failure leaves field reeling. Science. 2007;318:28–9. - PubMed
    1. Van Damme L, Govinden R, Mirembe FM, et al. Lack of effectiveness of cellulose sulfate gel for the prevention of vaginal HIV transmission. N Engl J Med. 2008;359:463–72. - PubMed
    1. Bolognesi N. AIDS gel’s failure calls prevention approach into question. Nat Med. 2007;13:230. - PubMed
    1. Honey K. Microbicide trial screeches to a halt. J Clin Invest. 2007;117:1116. - PMC - PubMed

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