Spontaneous preterm labor is associated with an increase in the proinflammatory signal transducer TLR4 receptor on maternal blood monocytes

<p>Abstract</p> <p>Background</p> <p>Localized inflammation and increased expression of TLR4 receptors within the uterus has been implicated in the pathogenesis of preterm labor. It remains unclear whether intrauterine inflammatory responses activate the maternal peripheral circulatory system. Therefore we determined whether increased TLR4 expression is present in the peripheral maternal white blood cells of women with spontaneous preterm labor.</p> <p>Methods</p> <p>This is a cross-sectional study of 41 preterm labor cases and 41 non-preterm controls. For each case and control sample, RNA was purified from white blood cells and TLR4 mRNA pool size was evaluated by quantitative PCR. Protein expression levels were determined by flow cytometry. Statistical evaluation using multiple linear regressions was used to determine any significant differences between the cases and controls. The purpose was to determine association prevalence of TLR4 levels and preterm labor.</p> <p>Results</p> <p>Adjusted mean TLR4 mRNA levels of 0.788 ± 0.037 (standard error) for preterm labor and 0.348 ± 0.038 for the corresponding pregnant control women were statistically significantly different <it>(P </it>= 0.002). Using the lower 95% confidence interval of the mean expression level in PTL subjects (0.7) as a cutoff value for elevated TLR4 mRNA levels, 25/41 (60.9%) of PTL patients expressed elevated TLR4 mRNA as compared to 0/41 (0%) in control subjects. The TLR4 receptor levels in the granulocyte fraction of white blood cells from preterm labor and pregnant controls were similar. However, TLR4⁺/CD14⁺monocytes were 2.3 times more frequent (70% vs. 30%) and TLR4 also had a 2.6-fold higher density (750 vs. 280 molecules per cell) in preterm labor women compared with pregnant controls. There was no difference in the levels of TLR4 in patients at term.</p> <p>Conclusions</p> <p>Patients with preterm labor exhibited elevated levels of CD14⁺maternal blood monocytes each bearing enhanced expression of TLR4, indicating that the peripheral circulatory system is activated in patients with preterm labor. Elevated leukocyte TLR4 levels may be a useful biomarker associated with preterm labor.</p

Protein expression profiles for CD55, CD14, and CD11b following addition of LPS (1 µg/ml) to THP-1 cells pretreated with or without 100 nM 1,25-D3 (72 h).

<p>A multiplexed fluorescence-based immunoblot approach was used to determine corresponding protein expression profiles from cells whose mRNA profiles are shown in Fig. 2. A, the temporal protein induction pattern of CD55, CD11b and β-actin following a LPS-challenge (without pretreatment). CD14 levels are not shown because the apparent 50-fold induction remained below the threshold of detection (nd). B, the relative band intensities for CD55 and CD11b, normalized to β-actin, are graphically presented as the average fold-change (relative to time zero) of three independent experiments (± SD). In some instances the SD are covered by the data point symbol. C and D, the multiplex immunoblot of LPS-induced protein expression in cells pretreated for 72 h with 1,25-D3 is shown in panel C; the fold induction is graphically represented in panel D as the average fold-change (relative to time zero) of three independent experiments (± SD). In some instances the SD are covered by the data point symbol. The apparent 100-fold induction of CD14 following 1,25-D3 pretreatment but before LPS (time = 0) was within the immunoblot detection limits.</p

NF-κB activation was required for LPS-induced CD55 expression in 1,25-D3 pretreated THP-1 monocytes.

<p>As illustrated under panel A, at the end of the 72 h pretreatment phase with 100 nM 1,25-D3, either vehicle or one of two NF-kB inhibitors [50 µM MG132 (MG) or 30 µm parthenolide (Pa)] was added and the incubation continued for an additional 1 h. Then LPS (1 µg/ml) or vehicle was added. Cells were ultimately harvested at 2 h (mRNA) or 24 h (protein) post addition of LPS or vehicle. Quantitative PCR was used to determine CD55 (A) and CD11b (D) mRNA levels assessed at 2 h. Differences between mRNA pool sizes were determined using a two tailed student’s t test. Asterisks (Fig. 5A) indicate statistical differences between indicated treatments; *P<0.05, **P<0.01. CD55 and CD11b protein expression was assessed at 24 h following a vehicle or LPS challenge by immunoblotting (B) and quantification (C and E), respectively. Because of the semi-qualitative nature of the protein determinations, a statistical analysis was not performed on these data. Values represent means +/− SD (n = 3).</p

CD55 expression was enhanced on the surface of essentially all THP-1 cells at 24 h, following addition of LPS to 1,25-D3 pretreated cells.

<p>THP-1 cells were grown on coverslips, pretreated with 100 nM 1,25-D3 for 72 h and then stimulated with either vehicle or LPS for 24 h, as indicated. Serial 0.5 µm optical sections were obtained by confocal microscopy (40 × magnification). A, sections containing either a central (internal Z-stack) or the apical region of the cells (cell surface Z-stack) and the entire 3D image of LPS treated cells are shown. White represents CD55 staining; nucleic acid staining was omitted for clarity. B, shows a graphical representation of mean (±SD) CD55 expression levels (normalized to nucleic acid content) for three independent experiments with two fields quantified per experiment (n = 6).</p

CD55 mRNA and protein expression was not altered during the early response phase (0–24 h) following a 1,25-D3 preincubation period of 72 h with human THP-1 monocytes.

<p>THP-1 cells were cultured in complete media in the absence or presence of 100 nM 1,25-D3 (added at time = 0) and harvested at the indicated times. A, CD55, CD14 and CD11b mRNA pool sizes were determined by quantitative PCR analysis using 18S rRNA as a reference. Fold-induction is expressed relative to vehicle treated controls. Values shown are the mean +/− SD of 3 independent experiments. In some instances, the SD bars are covered by the data point symbol. Shaded data points indicate statistical differences from control values. P<0.05 was considered significant. B, protein expression was analyzed in cells at various times following addition of 1,25-D3, using a fluorescence-based immunoblot approach. Corresponding controls showed no difference over the same time period.</p

Pretreatment with 1,25-D3 for 72 h sustained the subsequent LPS-induced expression of CD55 mRNA in THP-1 cells, compared to cells pretreated with vehicle for 72 h.

<p>THP-1 cells were first preincubated in complete culture media with vehicle or with 100 nM 1,25-D3. Seventy-two h later (t = 0 on A–C), either vehicle or 1 µg/ml LPS was added and the incubation continued for an additional 72 h. Cells were harvested at the indicated times and quantitative PCR (n = 3) was used to determine CD55 (A), CD14 (B) and CD11b (C) mRNA pool sizes using the absolute copy number method. Mean levels are expressed in arbitrary units normalized to 18S rRNA (± SD). Differences in mRNA pool size were determined using a two tailed student’s t test. Shaded data points indicate statistical differences from corresponding controls.</p

Epithelial Invasion by Escherichia coli Bearing Dr Fimbriae Is Controlled by Nitric Oxide-Regulated Expression of CD55

We previously reported that inhibition of nitric oxide (NO) increases the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr fimbria (Dr(+)). Epithelial binding and invasion by Dr(+) E. coli has also been shown to be dependent upon the expression level of the cellular receptor decay-accelerating factor (DAF; CD55). Here, we hypothesize that NO-related severity of infection could be mediated by changes in DAF expression and in the rate of epithelial invasion. The cellular basis of NO effects on epithelial invasion with Dr(+) E. coli was studied using Ishikawa endometrial carcinoma cells as an in vitro model of the human endometrial epithelium. Initially, we show that Ishikawa cells produce NO and express both NO synthase enzymes, NOS II and NOS III, and DAF protein. We next tested the abilities of both Dr(+) E. coli and a Dr(−) E. coli mutant to invade Ishikawa cells, and invasion was seen only with Dr(+) E. coli. Invasion by Dr(+) E. coli was decreased by elevated NO production and increased by NO inhibition. Elevated NO production significantly decreased DAF protein and mRNA expression in Ishikawa cells in a time- and dose-dependent manner. Here, we propose that in vitro invasion of an epithelial cell line is directly related to NO-regulated expression of DAF. The significance of NO-regulated receptor-ligand invasion is that it may represent a novel unrecognized phenomenon of epithelial defense against infection

Structure-Function Analysis of Decay-Accelerating Factor: Identification of Residues Important for Binding of the Escherichia coli Dr Adhesin and Complement Regulation

Decay-accelerating factor (DAF), a complement regulatory protein, also serves as a receptor for Dr adhesin-bearing Escherichia coli. The repeat three of DAF was shown to be important in Dr adhesin binding and complement regulation. However, Dr adhesins do not bind to red blood cells with the rare polymorphism of DAF, designated Dr(a(−)); these cells contain a point mutation (Ser165-Leu) in DAF repeat three. In addition, monoclonal antibody IH4 specific against repeat three was shown to block both Dr adhesin binding and complement regulatory functions of DAF. Therefore, to identify residues important in binding of Dr adhesin and IH4 and in regulating complement, we mutated 11 amino acids—predominantly those in close proximity to Ser165 to alanine—and expressed these mutations in Chinese hamster ovary cells. To map the mutations, we built a homology model of repeat three based on the poxvirus complement inhibitory protein, using the EXDIS, DIAMOD, and FANTOM programs. We show that perhaps Ser155, and not Ser165, is the key amino acid that interacts with the Dr adhesin and amino acids Gly159, Tyr160, and Leu162 and also aids in binding Dr adhesin. The IH4 binding epitope contains residues Phe148, Ser155, and L171. Residues Phe123 and Phe148 at the interface of repeat 2-3, and also Phe154 in the repeat three cavity, were important for complement regulation. Our results show that residues affecting the tested functions are located on the same loop (148 to 171), at the same surface of repeat three, and that the Dr adhesin-binding and complement regulatory epitopes of DAF appear to be distinct and are ≈20 Å apart

Pyelonephritogenic Diffusely Adhering Escherichia coli EC7372 Harboring Dr-II Adhesin Carries Classical Uropathogenic Virulence Genes and Promotes Cell Lysis and Apoptosis in Polarized Epithelial Caco-2/TC7 Cells

Diffusely adhering Escherichia coli (DAEC) strains expressing adhesins of the Afa/Dr family bind to epithelial cells in a diffuse adherence pattern by recognizing a common receptor, the decay-accelerating factor (CD55). Recently, a novel CD55-binding adhesin, named Dr-II, was identified from the pyelonephritogenic strain EC7372. In this report, we show that despite the low level of sequence identity between Dr-II and other members of the Afa/Dr family, EC7372 induces pathophysiological effects similar to those induced by other Afa/Dr DAEC strains on the polarized epithelial cell line Caco-2/TC7. Specifically, the Dr-II adhesin was sufficient to promote CD55 and CD66e clustering around adhering bacteria and apical cytoskeleton rearrangements. Unlike other Afa/Dr DAEC strains, EC7372 expresses a functional hemolysin that promotes a rapid cellular lysis. In addition, cell death by apoptosis or necrosis was observed in EC7372-infected Caco-2/TC7 cells, depending on infection time. Our results indicate that EC7372 harbors a pathogenicity island (PAI) similar to the one described for the pyelonephritogenic strain CFT073, which carries both hly and pap operons. Cumulatively, our findings indicate that strain EC7372 can be considered a prototype of a subclass of Afa/Dr DAEC isolates that have acquired a PAI harboring several classical uropathogenic virulence genes

Hydrophilic Domain II of Escherichia coli Dr Fimbriae Facilitates Cell Invasion

Uropathogenic and diarrheal Escherichia coli strains expressing adhesins of the Dr family bind to decay-accelerating factor, invade epithelial cells, preferentially infect children and pregnant women, and may be associated with chronic or recurrent infections. Thus far, no fimbrial domain(s) that facilitates cell invasion has been identified. We used alanine scanning mutagenesis to replace selected amino acids in hydrophilic domain II of the structural fimbrial subunit DraE and evaluated recombinant mutant DraE for attachment, invasion, and intracellular compartmentalization. The mutation of amino acids V28, T31, G33, Q34, T36, and P40 of DraE reduced or abolished HeLa cell invasion but did not affect attachment. Electron micrographs showed a stepwise entry and fusion of vacuoles containing Escherichia coli mutants T36A and Q34A or corresponding beads with lysosomes, whereas vacuoles with wild-type Dr adhesin showed no fusion. Mutants T31A and Q34A, which were deficient in invasion, appeared to display a reduced capacity for clustering decay-accelerating factor. Our findings suggest that hydrophilic domain II may be involved in cell entry. These data are consistent with the interpretation that in HeLa cells the binding and invasion phenotypes of Dr fimbriae may be separated