Distinct defensin profiles in Neisseria gonorrhoeae and Chlamydia trachomatis urethritis reveal novel epithelial cell-neutrophil interactions

Abstract

Defensins are key participants in mucosal innate defense. The varied antimicrobial activity and differential distribution of defensins at mucosal sites indicate that peptide repertoires are tailored to site-specific innate defense requirements. Nonetheless, few studies have investigated changes in peptide profiles and function after in vivo pathogen challenge. Here, we determined defensin profiles in urethral secretions of healthy men and men with Chlamydia trachomatis- and Neisseria gonorrhoeae-mediated urethritis by immunoblotting for the epithelial defensins HBD1, HBD2, and HD5 and the neutrophil defensins HNP1 to -3 (HNP1-3). HBD1 was not detectable in secretions, and HBD2 was only induced in a small proportion of the urethritis patients; however, HD5 and HNP1-3 were increased in C. trachomatis infection and significantly elevated in N. gonorrhoeae infection. When HNP1-3 levels were low, HD5 appeared mostly as the propeptide; however, when HNP1-3 levels were >10 microg/ml, HD5 was proteolytically processed, suggesting neutrophil proteases might contribute to HD5 processing. HD5 and HNP1-3 were bactericidal against C. trachomatis and N. gonorrhoeae, but HD5 activity was dependent upon N-terminal processing of the peptide. In vitro proteolysis of proHD5 by neutrophil proteases and analysis of urethral secretions by surface-enhanced laser desorption ionization substantiated that neutrophils contribute the key convertases for proHD5 in the urethra during these infections. This contrasts with the small intestine, where Paneth cells secrete both proHD5 and its processing enzyme, trypsin. In conclusion, we describe a unique defensin expression repertoire in response to inflammatory sexually transmitted infections and a novel host defense mechanism wherein epithelial cells collaborate with neutrophils to establish an antimicrobial barrier during infection.

FIG. 1.

Defensin profiles in urethral lavages from healthy men and men with STIs. Aliquots of urethral lavages were dialyzed against 5% acetic acid using a 3-kDa molecular weight cutoff membrane, subjected to AU-PAGE (for HD5 and HNP1-3) or SDS-Tricine-PAGE (for HBD1 and HBD2), electroblotted onto PSQ membrane, fixed, blocked, and probed with the appropriate rabbit polyclonal antibody, and defensin bands were visualized with alkaline phosphatase-conjugated polyclonal goat anti-rabbit antibodies and nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate substrate. Resulting band inten-sities were compared to the band intensities of standard peptides, and the approximate defensin concentrations in the various samples were deduced. Graphs were plotted as vertical boxes showing the 10th to 90th percentile, the median (solid line), the mean (dotted line), and the standard deviation of five different subjects per group. Statistical significance was calculated by the Kruskal-Wallis one-way analysis of variance. Note the larger y-axis scale for HNP. N, normal (healthy); NGU, nongonococcal nonchlamydial urethritis; CT, chlamydial urethritis; GC, gonococcal urethritis.

FIG. 2.

Representative immunoblots of urethral lavages probing for HD5 and HNP1 to -3. Samples were processed as described in the legend for Fig. 1. Equivalents of 100 μl of urethral lavage or 20 ng of standard peptide (proHD5 [aa 20 to 94], HD5 [aa 64 to 94], or HNP1) were subjected to AU-PAGE followed by immunoblotting for HD5 (A) or HNP1-3 (B). ST, peptide standard; N, normal (healthy); NGU, nongonococcal nonchlamydial urethritis; CT, chlamydial urethritis; GC, gonococcal urethritis. *, increased neutrophil counts in the urethral lavage sediment, which were paralleled by strongly elevated HNP concentrations of ≥10 μg/ml. Note the appearance of processed HD5 forms in samples with high HNP concentrations (by Fisher exact test, P < 0.05; see Table 1).

FIG. 3.

In vitro cleavage of proHD5 by neutrophil proteases. Experiments were conducted in 100 mM Tris-150 mM NaCl adjusted to various pHs. Purified enzymes were employed at a ratio of 1 molecule per 100 molecules of proHD5. After 2 h of incubation at 37°C, samples were placed on ice and diluted 1:2 in 5% acetic acid to prevent further proteolysis. Aliquots representing 100 ng proHD5 originally added to the reaction tubes were lyophilized and subjected to AU-PAGE followed by anti-HD5 immunoblotting. Co, control, no proteases; TRY, trypsin; PMN, neutrophil granule extract; EL, human neutrophil elastase; PR3, human neutrophil proteinase 3; LEU, leukozyme, human sputum leukocyte extract; ST, 50 ng recombinant proHD5 (aa 20 to 94) and HD5 (aa 56 to 94). Note: proHD5 tends to form multimeric aggregates that appear as additional slower migrating bands with lower intensity.

FIG. 4.

SELDI profiles of representative urethral lavages for each patient group. Note the different y-axis scales. HNP1 to -3 peptides produce a characteristic three-peak pattern around a mass ([m+z]/z) of 3,400, reflecting HNP1, HNP2, and HNP3 peptides (expected masses of 3,443, 3,371, and 3,486, respectively). For clarity, individual masses are not shown. See also Tables 2 and 3.

FIG. 5.

In vitro antibacterial activities of different HD5 forms with various N termini. Dose response relationships with recombinant proHD5 (aa 20 to 94), HD5 (aa 56 to 94), and HD5 (aa 63 to 94), tested at the same molarities, are shown. (A) For antigonococcal activity, defensins were mixed with 24-hour cultures of N. gonorrhoeae adjusted to ∼2 × 10⁶ CFU/ml in 70% RPMI supplemented with glucose and IsoVitaleX, and the number of CFU were determined after a 3-hour incubation. Values are expressed as change over the control at time zero. (B) For anti-C. trachomatis activity, defensins were added to infectious EB in SPG medium for 45 min prior to infection of Ishikawa cells. The inclusion-forming units (IFU)/well were enumerated 40 h after infection by immunohistochemistry using an anti-chlamydia lipopolysaccharide antibody. Data are expressed relative to the IFU/well resulting from infection with untreated chlamydia. Depicted are the means + standard deviations of three experiments.

FIG. 6.

Comparative defensin activity. (A) Various HD5 peptides were tested at a 3.3 μM concentration against E. coli cells under the same conditions as shown above for N. gonorrhoeae. (B) Activities of HNP1, HBD1, and HBD2 at a 3.3 μM concentration against N. gonorrhoeae. In panels A and B, the data depicted are the means minus standard deviations for three experiments; see Fig. 5 for details. (C) Dose-response curve for HNP1, HBD1 and HBD2, and HD5 (aa 56 to 94) for comparison against C. trachomatis. HBD1-40 and HBD1-44 are predominant native forms of HBD1 40 and 44 aminoacids in length, respectively. The assay was conducted as described in the legend for Fig. 5 with the exception that chlamydia were preincubated for 2 h with and without defensins prior to infection of Ishikawa cells. Depicted are the means of duplicates.

FIG. 7.

Antigonococcal activity of HD5 peptides generated from proHD5 through proteolytic cleavage by neutrophil proteases. Human neutrophil elastase (EL-HD5) and human neutrophil proteinase 3 (PR3-HD5) cleavage products were purified by reverse-phase HPLC and tested at a 5 μM final concentration. For assay details and data presentation, see the legend for Fig. 5A. Depicted are the means minus standard deviations of three experiments. (Insert) Two-microliter aliquots were taken from each sample of one experiment after completion of the incubation and subjected to AU-PAGE, followed by anti-HD5 immunoblotting. Lanes: 1, 50 ng recombinant proHD5 (aa 20 to 94) and HD5 (aa 56 to 94); 2, control at time zero (t0); 3, control after 3 h of incubation (t3); 4, untreated proHD5; 5, EL-generated HD5 peptides; 6, PR3-generated HD5 peptides.