<i>E. faecalis</i> MN1 secretes a factor responsible for inhibition of IL-8 production by HVECs in response to TSST-1.

<p>Transwell permeable supports (0.4 µm pore size) were used to determine the ability of <i>E. faecalis</i> MN1 secreted factors to inhibit TSST-1-induced IL-8 production by HVECs. HVECs were grown to confluency in the bottom wells and transwell permeable supports were added on the day of experimentation. <i>E. faecalis</i> MN1 (2×10⁷ CFU) was added to either the transwell (to assess secreted factors) or the bottom well (as a positive control) in the presence or absence of TSST-1 (100 µg/ml) and incubated for 6 h. In one condition, catalase enzyme (100 µg/ml) was added to break down hydrogen peroxide produced by the lactobacilli. Cell culture supernates were collected and analyzed by ELISA for IL-8 production. These results contain data averaged from three separate experiments, with 6 total replicates in each experimental condition. *All conditions with <i>E. faecalis</i> MN1 present demonstrated significantly lower IL-8 levels compared to the TSST-1 only control by individual Student's <i>t</i> tests (p<0.01).</p

<i>E. faecalis</i> MN1 inhibits the IL-8 response of HVECs to the staphylococcal superantigen TSST-1.

<p><i>E. faecalis</i> MN1 (1×10⁷ CFU) was incubated with HVECs in the absence and presence of TSST-1 (100 µg/ml) for 6 h. Cell culture supernates were collected and IL-8 was measured by ELISA. *Significantly higher than all other conditions and #significantly lower than medium only control by one-way ANOVA [<i>F</i>(3,29) = 251.2, <i>p</i><0.0001] and Tukey's post-hoc test. N = 3–6 replicates.</p

<i>E. faecalis</i> MN1 inhibits the IL-8 response of HVECs to vaginal pathogens.

<p>HVECs were incubated with <i>C. albicans</i> (2×10⁵ CFU/well), <i>G. vaginalis</i> (2×10⁶ CFU/well), or <i>N. gonorrhoeae</i> (2×10⁶ CFU/well) in the absence or presence of <i>E. faecalis</i> MN1 (8×10⁶ CFU/well) for 6 h and IL-8 production was measured by ELISA. *In each case, <i>E. faecalis</i> MN1 significantly inhibited the IL-8 response of the cells to the pathogen by individual Student's <i>t</i> tests (p<0.01). N = 3 replicates.</p

<i>E. faecalis</i> MN1 low molecular weight fraction inhibits TSST-1-induced T cell proliferation.

<p><i>E. faecalis</i> MN1 supernate was treated with ethanol (80% final concentration), insoluble material was removed by centrifugation, soluble material (referred to as the low molecular weight fraction) was concentrated 480 times relative to the original culture fluid, and dilutions incubated with human PBMCs and TSST-1 (1 µg/well) in a superantigenicity assay to assess the ability of secreted factors to inhibit TSST-1-induced T cell proliferation. The PBMC control shows the average CPMs for PBMCs alone and TSST-1 shows the average CPMs for TSST-1 alone. Treatments 48× to 0.0048× were significantly different from TSST-1 alone by individual Student's <i>t</i> tests (*p<0.001). Three independent wells for each treatment were assayed for cell viability, and viability in all cases was >95%.</p

<i>E. faecalis</i> MN1 secretes a small molecule responsible for inhibition of TSST-1-induced IL-8 production by HVECs.

<p>A) Eighty percent ethanol was used to precipitate high molecular weight factors from filter-sterilized <i>E. faecalis</i> MN1 supernate. Isolated factors were added to HVECs ± TSST-1 (100 µg/ml) for 6 h, and IL-8 was measured by ELISA. Only the 20 µl condition showed inhibition of TSST-1-induced IL-8 by Student's <i>t</i> test (p<0.001). B) Low molecular weight (unprecipitated) factors were concentrated by lyophilization, resuspended in PBS to the same concentration as the precipitated material, and incubated with cells as described above. Lower doses of unprecipitated material were used to minimize cytotoxicity to the HVECs. The 10 µl condition showed absolute inhibition of IL-8 production (*p<0.001), whereas the 1 µl condition actually enhanced IL-8 levels in response to TSST-1 (**p<0.001) by individual Student's <i>t</i> tests. These results are based on the average of two separate experiments, each containing three replicates per condition. C) Filter-sterilized <i>E. faecalis</i> MN1 supernate was separated into different molecular weight fractions using Microcon centrifugal filters and incubated with HVECs +/− TSST-1 (100 µg/ml) for 6 h. Inhibition of TSST-1-induced IL-8 production is maintained in all fractions (<3 kDa, <10 kDa, <30 kDa) when the cells were incubated with 20 µl of material (*p<0.005); a higher level of IL-8 was detected when cells were incubated with smaller amounts of the <30 kDa fraction (**p<0.05) by individual Student's <i>t</i> tests. These data are from a single experiment carried out in triplicate. D) Filter-sterilized <i>E. faecalis</i> MN1 supernate was treated with DNase, RNase, or protease prior to incubation with TSST-1 (100 µg/ml) on HVECs for 6 h. None of the treatments affected the ability of the secreted factor to inhibit TSST-1-induced IL-8 production from the cells by individual Student's <i>t</i> tests (*p<0.001). These data are the average of two experiments, each done in triplicate.</p

<i>E. faecalis</i> MN1 supernate inhibits TSST-1-induced T cell proliferation.

<p><i>E. faecalis</i> MN1 supernate was incubated with human PBMCs and TSST-1 (1 µg/well) in a superantigenicity assay to assess the ability of secreted factors to inhibit TSST-1-induced T cell proliferation. The two highest doses of supernate (20 and 2 µl per well) significantly inhibited T cell proliferation due to TSST-1 by individual Student's <i>t</i> tests (*p<0.001). The 0 µl control shows the average CPMs for PBMCs with and without TSST-1.</p

<i>E. faecalis</i> MN1 makes low levels of hydrogen peroxide.

<p><i>E. faecalis</i> MN1 and <i>L. crispatus</i> ATCC 33197 were grown overnight in KSFM tissue culture medium. Hydrogen peroxide production was measured using a H₂O₂ colorimetric detection assay kit.</p

Sensitivity of MN detection with PolyT length and dye variants.

<p>Each of the indicated probes was combined with the various indicated concentrations of MN (diluted in buffer) and incubated at 37°C for 1 hour and then fluorescence was measured. Fluorescence values are normalized to those of control samples in which no nuclease was included. Fluorescence values for the lowest MN concentrations are also plotted with a different scale for better differentiation of the lower values (inset). Error bars indicate standard deviations of triplicate measurements.</p

Nuclease activity in culture supernatants of <i>S</i>. <i>aureus</i>, <i>S</i>. <i>epidermidis</i> and <i>S</i>. <i>lugdunensis</i>.

<p>An overnight culture supernatant of each species was incubated with the 4mer PolyT ATTO probe for 1 hour at 37°C and fluorescence was measured. Fluorescence values are normalized to that of a control sample in which buffer was substituted for culture supernatant; note normalization values above each bar.</p

Inactivation of MN inhibitory activity with a heat treatment step.

<p>Human serum and plasma that was spiked with calcium chloride and the indicated amounts of MN was either heated to 90°C or incubated at 4°C, for 20 minutes (A). Unheated samples and supernatants of heated samples were then incubated with the 11mer PolyT FAM probe and fluorescence was measured. Note the clear differences between the heated and unheated samples with MN concentrations below 10⁻² units/μl (panel in A). Error bars indicate standard deviations of triplicate measurements. The incubation time needed to inactivate inhibitory antibodies in plasma at 90 and 100°C was determined as follows. Human plasma, spiked with 10⁻⁵ units/μl MN and 10 mM calcium chloride, was heated to 90°C (upper panel in B) or 100°C (lower panel in B) for indicated amounts of time, centrifuged, and supernatants were incubated with the 11mer polyT FAM probe. Probe incubated with buffer only was included as control. Error bars indicate standard deviations of triplicate measurements.</p