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Introduction: Assessment of specific mRNAs in human samples is useful in characterizing disease. However, mRNA in human stool has been understudied. Results: Compared to controls, infected stools showed increased transcripts of IL-1β, IL-8 and calprotectin. mRNA and protein concentrations correlated for IL-8, but not for calprotectin. Discussion: Stool mRNA quantification offers a potentially useful, noninvasive way to assess inflammation in the gastrointestinal tract, and may be more sensitive than EIA. Methods: We purified fecal RNA from 46 children infected with Campylobacter jejuni, Escherichia coli O157:H7, Salmonella spp. or Shigella sonnei and 26 controls and compared the proportions of IL-1β, IL-8, osteoprotegerin and calprotectin mRNA between groups using qRT-PCR. We determined the concentrations of calprotectin, IL-8 and osteoprotegerin by enzyme immunoassays in cognate specimens.
Assessments of specific messenger RNAs in human specimens, including blood,1 urine2 and tissue3 are of great utility in characterizing health and disease states. Fecal mRNA has been underexplored as an analyte, probably because of reservations regarding its feasibility. However, recent reports4,5 demonstrate that stool mRNA isolation and analysis is reproducible and useful in characterizing a wide array of host intestinal responses in children and adults. If fecal mRNA reflects pathophysiology within the digestive system, its analysis could open new vistas on gut biology in health and disease, and supplant protein determinations such as enzyme immunoassays (EIAs).
To test the feasibility of mRNA assessment in human intestinal infections, we measured the relative (to GAPDH mRNA) abundances of selected mRNAs in stool from patients infected with Campylobacter jejuni, Escherichia coli O157:H7, Salmonella and Shigella. The mRNAs correspond to a panel of pro-inflammatory host proteins (IL-1β, IL-6, IL-8, calprotectin and osteoprotegerin (OPG)), which are found in excess in the stool in infectious or inflammatory diarrhea by EIA, or have been found to be associated with the intestinal immune response to infection.6–11 We also compared the resulting transcript ratios to protein concentrations as determined by EIAs for IL-8, calprotectin and OPG.
Patient groups and control groups did not differ significantly by age, gender or race (Table 1).
IL-8, IL-1β and calprotectin relative transcript abundances were significantly elevated in the stools of infected patients (all pathogens) compared to the control group (Fig. 1A–C, individual and cumulative p values all < 0.0001). The relative abundances of IL-6 and OPG mRNA were not detectable in either patients or controls (the limit of sensitivity was 10 copies of mRNA per reaction tube) (data not shown). For all mRNA species, purified host control mRNA (positive controls) amplified effectively, while water (negative controls) did not. No amplicons were produced when samples were amplified without the initial RT step.
IL-8 concentrations were significantly higher in the stools of patients with infectious colitis than in controls (Fig. 1D, individual and cumulative p values all < 0.0001), but calprotectin concentrations were not (cumulative p value = 0.48). OPG concentrations were low in both cases and controls (median 10.4, range 2.1–143.3 and median 3.4, range 2.2–5.3, respectively, all values pmol/L) (cumulative p value = 0.17).
These results extend our previous work on neonatal stool, and the work of others on stools from subjects with bacterial enteric infections, by portraying the ability to characterize the host inflammatory response to these pathogens using fecal mRNA analysis. We also demonstrate the variable correlation of fecal mRNA abundance with the cognate protein. This disparity was most notable for calprotectin mRNA abundances and protein antigen.
We cannot state if these results are specific for infectious injuries to the gut, and/or the extent to which specific mRNAs might be found in non-infectious gastrointestinal inflammation. Stool cytokines (usually detected via immunoassays) are often elevated in inflammatory bowel disease,14,15 so it would not be surprising if corresponding fecal mRNAs are also present in these and other inflammatory disorders and elevated compared to controls. It is also possible that additional colonic (e.g., diverticulitis, ischemic colitis) and more proximal disorders (e.g., celiac disease) might also be characterized by measuring stool mRNAs.
Target stability is a major consideration in any assay, and analyses of molecules in stool present particular concern. We have noticed proteolysis in stools using spiked cytokines and protein immunoblots (data not shown), and it is possible that host or microbial proteases render protein molecules undetectable by EIA, either because of degradation or denaturation of target. There could be additional reasons for non-detection of calprotectin by EIA beyond proteolysis. The EIA kit we employed uses antibodies to the calprotectin complex (a heterodimer of S100A8 and S100A9 subunits), while the primers used to detect calprotectin via qRT-PCR were for only the S100A8 moiety. Denaturation of the protein or dissociation of the subunits could hinder EIA detection if the target epitopes are changed or separated.
We previously demonstrated the relative stability of host mRNA in neonatal stool.5 Even if RNA degradation does occur in stool, as long as the mRNA of interest and the GAPDH transcripts degrade at the same rates, and sufficient non-degraded transcript (the calprotectin and GAPDH primers produce RNA amplicons that are 107 and 122 base pairs, respectively) remains, ratios can be determined. There are two additional advantages to determining mRNA abundance in stool, compared to assays of proteins. First, mRNAs are normalized to a host housekeeping transcript, and are independent of fecal water content. This is particularly important in diarrhea, where dilutions of target protein could yield falsely low values in patients compared to controls with solid stools. Second, mRNA detection is more economical. By our calculations, the reagent cost of performing duplicate EIA determinations on a specimen is about twice that of assessing relative mRNA abundances. Indeed, the per-assay cost differences are probably greater when considering that the fixed cost of the RNA preparation, which accounts for about half the cost of a single mRNA abundance determination, can be offset by the ability to measure multiple additional transcripts. Such single analyte multidimensional utility increases the ability to profile digestive organ response with parsimonious use of reagents.
In summary, we extend previous work by demonstrating that human stool mRNAs are quantifiable analytes in infectious colitis. mRNAs from several proteins important in inflammatory cascades are consistently elevated in the stools of patients with acute infectious colitis, and such assessments provide greater sensitivity than immunoassays. To the extent we tested, there is no evidence that EIAs better detect inflammation than mRNA abundances, and it appears likely that relative mRNA abundances offer equivalent or improved sensitivity for characterizing host response in acute bacterial enteric infections.
Forty-six stools containing C. jejuni, E. coli O157:H7, Salmonella and Shigella were obtained primarily from a Seattle pediatric emergency department study of diarrhea etiology.12 We also used additional age-matched (to the controls from the emergency department study) specimens of convenience from additional infected children that were obtained in a multistate prospective study of E. coli O157:H7 infections.13 Stools were frozen since collection at −80°C. All specimens were collected with the approval of the Human Research Protection Office at Washington University, Seattle Children's Hospital and equivalent bodies at collaborating institutions. Specimen details, pathogens (if any) that they contained, and the subjects from which they were obtained are provided in Table 1.
For the majority of the mRNA analyses (IL-6, IL-8, IL-1β, EGF and calprotectin), all 46 cases and 23 controls were used. For a subset of these samples, 19 cases and 12 age matched controls, were subjected to protein analysis by EIA, and for mRNA analysis of OPG. The OPG assays were limited in number because of sample volume limitations. Cases without controls were selected to assure that age and gender did not differ significantly from those with matched controls. When comparing controls with cases, the results from all controls were considered as a single group and also as a matched group for statistical analyses.
The Wilcoxon rank-sum test was used to determine the significance of differences for age, gender and race for patients and control groups, as well as to determine the significance of difference between patient and control subjects' relative mRNA abundances and protein concentrations. p values <0.05 (two-tailed) were considered statistically significant.
EIAs were used to quantify IL-8 (R&D Systems, Inc.; Minneapolis, MN-PID D8000C), calprotectin (Hycult Biotechnology, Netherlands-PID HK325) and OPG (ALPCO Immunoassays, Salem, NH-BI-20402) using stools diluted 1:10 in PBS, then centrifuged. Supernatants were processed according to the manufacturer's instructions (IL-8, R&D Systems, Inc., Minneapolis, MN; Calprotectin, Hycult Biotechnology, Uden, The Netherlands). For OPG, stools were diluted 1:5 or 1:10 in HBSS to obtain a total minimum volume of 300 µl, centrifuged and supernatants processed according to the manufacturer's instructions (OPG, ALPCO Immunoassays, Salem, NH). Absorbances for IL-8 and calprotectin were determined on a VersaMax plate reader (Molecular Devices, Sunnyvale, CA, USA) and for OPG using a BIO-RAD Benchmark microplate reader (BIO RAD, Hercules, CA). The limits of sensitivity for these assays are 0.60 pmol/L, 0.44 pmol/L and 0.14 pmol/L, respectively.
RNA was isolated from stool samples as described5 and DNase digested prior to elution from the silica column. We used quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to measure the ratios of various host mRNAs. Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), IL-6, IL-8, IL-1β, OPG and S100A8 (a calprotectin subunit) were obtained from Applied Biosystems, Inc., Foster City, CA. [Product numbers: Hs99999905_m1 (GAPDH), Hs99999032_m1 (IL-6), Hs00171403_m1 (IL-8), Hs01555410_m1 (IL-1β), Hs00374264_g1 (S100A8), Hs00900360_m1 (OPG-3′ end of transcript) and Hs00171068_m1 (OPG-5′ end of transcript)]. The priming sites flanked introns, so as to amplify RNA and not DNA.
Ten µL reactions were prepared using 3 µL of RNA (corresponding to ~200 ng RNA per reaction), 5 µL TaqMan 2X Environmental Master Mix, 0.5 µL 20X primer mix, 0.1 µL SuperScript III Reverse Transcriptase, 0.2 µL RNase inhibitor and 1.2 µL RNase-free H2O (all PCR reagents from Applied Biosystems, Inc.). RT-PCR was performed in a 7500 Fast Real Time PCR System (Applied Biosystems, Inc.) using the following cycling conditions: 48°C for 30 minutes, followed by 95°C for 10 minutes, then 40 cycles of 95°C for 15 seconds, 60°C for 15 seconds and 72°C for 60 seconds. Copy numbers for each transcript in each sample were calculated using 7500 Fast Real-Time PCR System Sequence Detection Software v. 1.3.1 (Applied Biosystems, Inc.). Positive controls consisted of purified total human control RNA (Applied Biosystems, Inc.) and negative controls consisted of RNase-free H2O. Positive controls amplified effectively with each primer and negative controls did not amplify above threshold values for any primer pair. In a subset of samples, we attempted to make amplicons with the initial RT set omitted to confirm completeness of DNA digestion.
This research has been supported by NIH grants NIDDK T32 DK077653-17, UH2 AIO83265, and the Doris Duke Clinical Research Fellowship Program. Specimens were obtained using resources from grants NIH DK 52081 and USDA 2002-35212-14257.
Previously published online: www.landesbioscience.com/journals/gutmicrobes/article/13004