Trappin-2/Elafin Modulate Innate Immune Responses of Human Endometrial Epithelial Cells to PolyI∶C

BACKGROUND: Upon viral recognition, innate and adaptive antiviral immune responses are initiated by genital epithelial cells (ECs) to eradicate or contain viral infection. Such responses, however, are often accompanied by inflammation that contributes to acquisition and progression of sexually transmitted infections (STIs). Hence, interventions/factors enhancing antiviral protection while reducing inflammation may prove beneficial in controlling the spread of STIs. Serine antiprotease trappin-2 (Tr) and its cleaved form, elafin (E), are alarm antimicrobials secreted by multiple cells, including genital epithelia. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated whether and how each Tr and E (Tr/E) contribute to antiviral defenses against a synthetic mimic of viral dsRNA, polyinosine-polycytidylic acid (polyI:C) and vesicular stomatitis virus. We show that delivery of a replication-deficient adenovector expressing Tr gene (Ad/Tr) to human endometrial epithelial cells, HEC-1A, resulted in secretion of functional Tr, whereas both Tr/E were detected in response to polyI:C. Moreover, Tr/E were found to significantly reduce viral replication by either acting directly on virus or through enhancing polyI:C-driven antiviral protection. The latter was associated with reduced levels of pro-inflammatory factors IL-8, IL-6, TNFα, lowered expression of RIG-I, MDA5 and attenuated NF-κB activation. Interestingly, enhanced polyI:C-driven antiviral protection of HEC-Ad/Tr cells was partially mediated through IRF3 activation, but not associated with higher induction of IFNβ, suggesting multiple antiviral mechanisms of Tr/E and the involvement of alternative factors or pathways. CONCLUSIONS AND SIGNIFICANCE: This is the first evidence of both Tr/E altering viral binding/entry, innate recognition and mounting of antiviral and inflammatory responses in genital ECs that could have significant implications for homeostasis of the female genital tract

Anti-HIV-1 activity of elafin depends on its nuclear localization and altered innate immune activation in female genital epithelial cells.

Elafin (E) and its precursor trappin-2 (Tr) are alarm antiproteases with antimicrobial and immunomodulatory activities. Tr and E (Tr/E) have been associated with HIV-1 resistance. We recently showed that Tr/E reduced IL-8 secretion and NF-κB activation in response to a mimic of viral dsRNA and contributed to anti-HIV activity of cervicovaginal lavage fluid (CVL) of HIV-resistant (HIV-R) commercial sex workers (CSWs). Additionally, Tr, and more so E, were found to inhibit attachment/entry and transcytosis of HIV-1 in human endometrial HEC-1A cells, acting through virus or cells. Given their immunomodulatory activity, we hypothesized that Tr/E could exert anti-HIV-1 activity at multiple levels. Here, using tagged and untagged Tr/E proteins, we comparatively evaluated their protease inhibitory, anti-HIV-1, and immunomodulatory activities, and cellular distribution. E appeared to function as an autocrine/paracrine factor in HEC-1A cells, and anti-HIV-1 activity of E depended on its unmodified N-terminus and altered cellular innate activation, but not its antiprotease activity. Specifically, exogenously added N-terminus-unmodified E was able to enter the nucleus and to reduce viral attachment/entry and transcytosis, preferentially affecting R5-HIV-1(ADA), but not X4-HIV-1(IIIB). Further, anti-HIV-1 activity of E was associated with significantly decreased HIV-1-triggered IL-8 release, attenuated NF-κB/p65 nuclear translocation, and significantly modulated mRNA expression of innate sensors TLR3 and RIG-I in HEC-1A cells. Most importantly, we found that elevated Tr/E in CVLs of HIV-R CSWs were associated with lower mRNA levels of TLRs 2, 3, 4 and RIG-I in the genital ECs from this cohort, suggesting a link between Tr/E, HIV-1 resistance and modulated innate viral recognition in the female genital mucosa. Collectively, our data indicate that unmodified N-terminus is critical for intranuclear localization and anti-HIV-1 activity of E. We also propose that E-mediated altered cellular innate activation most likely contributes to the HIV-R phenotype of these subjects

Enhanced polyI∶C-driven antiviral protection in Ad/Tr-cells is associated with hyperactivation of IRF3.

<p>(<i>A</i>) Western blots and their analyses of phosphorylated IRF3, total IRF3, and GAPDH proteins were performed from whole-cell extracts of HEC-Ad cells that were either left untreated or treated with 25 µg/ml polyI∶C during indicated time points. (<i>B</i>) Immunofluorescence analysis of IRF3 nuclear translocation following either medium alone or polyI∶C 25 µg/ml treatment for 4 h. Representative staining is shown for IRF3 (green), nuclear stain (PI) (red), and composite (yellow) at magnification 2520×. The data are representative of three independent experiments with similar results.</p

Predominant sTLR2 polypeptides profiles in breast milk.

<p>Predominant sTLR2 polypeptide profiles found in multiple breast milk (BM) samples using Western and Native blot analysis. (<b>A</b>) BM samples (10 µg total milk protein) with commercial rsTLR2 were evaluated by Western blot analysis with N-17 pAb and 4 mAb (sc-52909, T2.5, TL2.3, TL2.1). pAb N-17 detected commercial rsTLR2 as well as multiple bands in BM; the predominant BM forms were ∼83 kDa and ∼38 kDa sTLR2 forms. In contrast, mAbs did not detect the commercial rsTLR2. In BM mAbs detected the ∼83 kD band, as well as a unique ∼26 kDa sTLR2 form, which was not detected with the N-17 pAb. (<b>B</b>) N-17 pAb and T2.5 mAb were used in Native blot analysis of two BM samples. N-17 pAb detected two large proteins (arrow 1 & 2), while T2.5 mAb detected 3 proteins (arrow 1, 2 & 3). A representative data set from three experiments is shown.</p

Specificity of anti-sTLR2 antibodies.

<p>The specificity of anti-sTLR2 antibodies was confirmed using a peptide competition assay and CBA. (A) N-17 was preincubated without (-P) or with (+P) 5× molar excess of peptide (N-17P) prior to immunoblotting. Pre-incubation with excess peptide markedly reduced both the ∼83 kDa and ∼38 kDa isoforms of sTLR2. Results representative of breast milk (BM) samples from different donors tested. (B) Schematic of cytometric bead array shows that beads coated with T2.5 mAb pulled natural sTLR2 out of milk and were detected with phycoerythrin (PE) labeled N-17 detection antibody. (C) CBA analysis of commercial rsTLR2 and BM dilution clearly shows system can detect natural sTLR2 but cannot detect commercial rsTLR2. A representative data set from triplicate experiments is shown.</p

Ad/Tr-cells respond to polyI∶C treatment with reduced production of IFNβ.

<p>HEC-1A cells were either treated with medium alone (UT) or with MOI 50 of Ad/dl or Ad/Tr and incubated for 6–24 h in presence of media or 25 µg/ml of polyI∶C. (<i>A</i>) At 6 h post treatment, IFNβ mRNA from total RNA was determined by real-time quantitative RT-PCR. Values are normalized to a housekeeping gene 18S in the same sample and presented as fold induction over UT cells in absence of polyI∶C treatment. (<i>B</i>) At 24 h of polyI∶C stimulation, supernatants were tested for IFNβ expression by ELISA. Data are representative of at least two independent experiments performed in triplicate and expressed as the mean ± SD, shown in pg/ml. Statistical analysis was performed using Student's <i>t</i> test with * representing significant difference between the groups, p<0.05.</p

Characteristics of Healthy HIV-uninfected Women Participants.

<p>Characteristics of Healthy HIV-uninfected Women Participants.</p

sTLR2 significantly inhibits HIV-1 infection in reporter assay.

<p>(A) MTT assay indicates that HIV-uninfected breast milk (BM) was not toxic to TZM-bl cells. (B) HIV-uninfected BM was incubated with either N-17 pAb or T2.5 mAb (200 before R5 HIV-1 (200 TCID₅₀) and then placed on TZM-bl cells. T20, 2F5IgG, and 1∶100 BM significantly inhibited infection (<i>P<0.001)</i>. A significant increase (<i>P<0.001, 0.01,</i> respectively<i>)</i> in HIV-1 infection was shown when sTLR2-specific N-17 or T2.5 Ab were pre-incubated with BM. N-17 pAb and T2.5 mAb alone and isotype control (200 ng/mL) did not inhibit HIV-1 infection. (C) rsTLR2+/− sCD14 or pooled HIV-uninfected mock-D or sTLR2-D BM (described in materials and methods) were incubated with R5 HIV-1 (200 TCID₅₀) before addition to TZM-bl cells for 48 hours. A significant decrease (<i>P<0.001</i>) in HIV infection was observed in cells exposed to mock-D BM. However, a significant increase (<i>P<0.001</i>) in HIV infection was detected with sTLR2-depleted BM. (D) rsTLR2+/− sCD14, mock-D, or sTLR2-D was incubated with cells for 1 hour at 37°C, washed with PBS, and then exposed to R5 HIV-1 (200 TCID₅₀) did not alter HIV-1 infection. <i>** P<0.01,</i> ***<i>P<0.001</i>. Errors bars, SEM. A representative data set from four experiments is shown.</p

sTLR2-mediated augmentation of pro-inflammatory cytokines during bacterial lipoprotein exposure.

<p>(A) Immunodepletion of ∼38 kDa sTLR2 in breast milk (BM) described in materials and methods. Quantitative analysis using TLR2 ELISA indicated a significant (<i>P<0.05)</i> decrease in sTLR2-depleted (sTLR2-D) compared to mock-D breast milk (BM). Western blot analysis revealed that ∼38 kDa and ∼83 kDa sTLR2 were markedly reduced compared to mock-D BM. (B-D) 500 ng/mL Pam₃CSK₄ was incubated with media or BM that was either mock-D or sTLR2-D for 1 hr at 37°C before being placed on cells. Supernatants were collected for IL-8 ELISA after 18 hours. Results represent (B) U937, (C) HEK293-TLR2 and (D) HEK293. (E) Commercial rsTLR2 was used at varying concentration with or without sCD14 and showed no inhibition of IL-8 or (F) IL-6 production in HEK293-TLR2 cells. Significant increases in pro-inflammatory cytokine, IL-8, was observed in sTLR2-D compared to mock-D BM. *<i>P<0.05,</i> **<i>P<0.01</i>, ***<i>P<0.001</i>. Errors bars, SEM. A representative data set from triplicate experiments is shown.</p