doi:10.1111/codi.12503
Original article
Correlations between colonic crypt mucin chemotype, inflammatory grade and Desulfovibrio species in ulcerative colitis Balfe*†, N. Bambury*†, A. Lavelle*†, A. Maguire‡, N. G. Docherty§, G. Lennon*†, A. J. C. Coffey¶, D. C. Winter*†, K. Sheahan†‡ and P. R. O’Connell*† *School of Medicine and Medical Sciences, University College Dublin, Dublin, Ireland, †Centre for Colorectal Disease, St Vincent’s University Hospital, Dublin, Ireland, ‡Department of Histopathology, St Vincent’s University Hospital, Dublin, Ireland, §Department of Physiology, School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland and ¶Graduate Entry Medical School, University Hospital Limerick, University of Limerick, Limerick, Ireland Received 12 June 2013; accepted 2 October 2013; Accepted Article online 18 December 2013
Abstract Aim The colonic mucus gel layer is composed of mucins that may be sulphated or sialyated. Sulphated mucins predominate in health while in ulcerative colitis (UC) sulphation is reduced. These differences result directly from inflammatory events. It may also be hypothesized that they arise in part from alterations in the colonic microbiota, particularly changes in the burden of sulphated mucin-metabolizing species, such as Desulfovibrio (DSV) bacteria. The aim of this study was to correlate colonic mucin chemotypes and inflammatory scores in health and UC and relate these changes to changes in the colonization of colonic crypts by DSV. Method Paired colonic biopsies from 34 healthy controls (HC) and 19 patients with active UC were collected for the purpose of parallel histological and microbiological assessment. High-iron diamine and Alcian blue staining and haematoxylin and eosin of mucosal biopsy specimens were used to assess histological changes within the clinical spectrum of UC. Quantitative real-time polymerase chain reaction analysis was employed to determine the total and DSV copy number within the colonic crypts.
Results Compared with HC, the mucin chemotype in UC was less sulphated and inversely correlated with the degree of mucosal inflammation. A weak but significant negative correlation was found between the abundance of sulphated mucins and DSV burden. Conclusion Mucin composition strongly correlates with the degree of mucosal inflammation, and to a lesser extent with DSV burden. These data suggest that mucin chemotype and DSV burden are linked phenomena and highlight the need to consider changes in mucin chemotype in the setting of microbial dysbiosis occurring within the colitic colon. Keywords Ulcerative colitis, mucin chemotype, Desulfovibrio What does this paper add to the literature? Decreased sulphation of mucins has been associated with inflammation in ulcerative colitis. Currently there are few data describing the relationship between microbial species and changes in mucin chemotype. This study validates previous findings and presents evidence of changes in mucin chemotype occurring in tandem with coherent changes in the microbiota within crypt niches.
Introduction Correspondence to: Prof. P. Ronan O’Connell, Surgical Professorial Unit, St Vincent’s University Hospital, Elm Park, Dublin 4, Ireland E-mail: [email protected] Portions of this work were presented at the ESCP 2011 meeting in Copenhagen, Denmark and the SARS 2011 meeting in Dublin, Ireland and have been published in abstract form in Colorectal Disease 13 (Suppl 6), 2011 and British Journal of Surgery 99 (Suppl 4), 2012.
The mucus gel layer (MGL) in the colon is an important component of the innate immune system. It serves both as a barrier protecting colonic mucosa from harmful substances in the lumen and as an interface between the host epithelium and commensal bacteria present in the lumen [1]. In ulcerative colitis (UC) the MGL is quantitatively and qualitatively different from that in
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health, attributed to premature discharge of mucins from goblet cells [2,3]. The effects of changes in the glycoprotein and mucin content [4,5] on MGL function, and their relationship with the pathogenesis of UC, remain uncertain; however, both are likely to be important in the complex interplay between the colonic microbiota and epithelial immune responses. Mucin is a critical component of the MGL; it consists of a peptide backbone with alternating glycosylated and nonglycosylated domains [6]. Mucins can be classified as neutral or acidic, with the acidic mucins being further classified as sialyated or sulphated [7]. Sialyated mucins are converted to mature sulphated forms through posttranslational modification. Typically, the mucins expressed in the healthy human colon are predominantly sulphated [1,5]; however, in UC a predominance of unsulphated mucin (sialyated mucin type) has been observed [5,8,9] and the MGL is thinner due to a reduction in the volume of mucin present [3]. Type 2 mucin (MUC2) is the predominant mucin secreted by the colonic epithelium. A qualitative change in MUC2 structure occurs during acute inflammation, with an aberrant glycan profile occurring in patients with active UC [8,10]. These alterations in mucin type, together with a reduction in MGL thickness, may facilitate access of luminal bacteria or bacterial products to the underlying epithelium. Such changes are thought to be influenced by bacterial mucinases, including peptidases, glycosidases, sulphatases and esterases, all of which are increased in the colitic colon [9]. In health there is a constant equilibrium between synthesis of colonic mucins and breakdown by resident bacteria that possess the enzymes necessary to utilize colonic mucins as an energy source [11]. In health, colonic mucins are highly sulphated and this may act as a bacteriostatic factor providing resistance to enzymatic degradation [12]. Sulphation of mucins is also related to bacterial load, as evidenced by increased ratios of sulphated mucins in the mucosa of ileal pouch reservoirs, correlating with an increased luminal bacterial load [13]. Colonic crypts, which are laden with mucins, provide a niche for host–bacterial interaction [11,13]. Previously our group reported the use of laser capture microdissection (LCM) combined with quantitative real-time polymerase chain reaction (qRT-PCR) to study bacterial colonization of the colonic MGL. A decrease in total bacterial copy number and increase in Desulfovibrio (DSV) burden was noted at sites of severe inflammation and mucosal ulceration in specimens from patients with acute UC [13,14]. Desulfovibrio are common gut commensals and are the most abundant genus of sulphate-reducing bacteria (SRB) in the human colon [15]. These bacteria are
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capable of metabolizing colonic sulphomucin-derived sulphate, utilizing the sulphate for metabolism and in so doing producing hydrogen sulphide (H2S) gas which has potential genotoxic effects [12]. Whilst controversial, a body of evidence exists in the literature implicating SRB, DSV and H2S in UC-associated inflammation [12,16]. Therefore it could be hypothesized that the changes in mucin chemotype characteristic of UC may be driven not only by inflammation but also by changes in the abundance of SRB such as DSV. The aim of the present study was to quantify the expression of sulphated mucin chemotypes in health and acutely active UC and to investigate possible relationships between the mucin chemotype composition and the presence of DSV at the level of the preserved crypt structures within the colitic colon.
Method Patient recruitment
The experimental protocol was approved by the St Vincent’s University Hospital Ethics Committee, and written informed consent was obtained prior to sample collection. Enrolled subjects were over 18 years of age. Healthy controls (HC) had no history of inflammatory bowel disease (IBD) and were undergoing routine day case colonoscopy for polyp surveillance. Patients with acute UC (AUC) were recruited at the time of colectomy for chronic colitis refractory to medical treatment, acute severe colitis unresponsive to infliximab or fulminant colitis requiring emergency surgery. Subjects were excluded if they had received antibiotic treatment in the preceding 6 weeks, with the exception of patients with AUC who all received prophylactic broad-spectrum antibiotics at induction of anaesthesia. All HC had received bowel preparation with polyethylene glycol and sodium picosulphate prior to colonoscopy and sample collection. A total of 34 HC and 19 AUC patients were recruited to the study. The patient demographics for each cohort are given in Table 1. Sample collection
All samples were collected from HC subjects at the time of colonoscopy. Following successful caecal intubation, duplicate mucosal biopsies were taken at each of four levels (right colon, transverse colon, left colon and rectum) using a Radial Jaw3 biopsy forceps (Boston Scientific, Natick, Massachusetts, USA) (Fig. 1). Likewise four duplicate specimens were collected immediately following specimen delivery at the time of surgery in the AUC cohort. Biopsy specimens from both cohorts were
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Table 1 Summary of patient demographics in healthy controls and acute ulcerative colitis (UC).
Gender (male, female) Age (years) Smoking (n) Non-smoker Former smoker Current smoker Unknown Years since diagnosis Disease activity Mayo score Antibiotic treatment with previous 6 months*
Healthy controls
Acute UC
16, 18 55.5 [23–82]
9, 12 42.4 [23–83]
(12) (2) (3) (17) n/a
(6) (5) (0) (8) 8.5 [1–38]
n/a n/a
10 [8–11] (1)
Values given are mean [range], and (number of patients). *Refers to patients in receipt of antibiotic treatment 6 months prior undergoing the procedure but not within the 6-week exclusion period for this study.
processed in the same manner. One biopsy from each region sampled was placed in 10% neutral buffered formalin for a minimum of 24 h. Fixed biopsies were then paraffin embedded and used for histological analysis. The second biopsy specimen was placed in Tissue Tek OCT medium (Sakura Finetek Europe, Alphen aan den Rijn, The Netherlands), snap-frozen in liquid nitrogen and stored at 80°C for molecular analysis. Mucin histochemistry and histopathological analysis
To identify and differentiate between sulpho- and sialomucins, biopsy sections were stained with high-iron diamine and Alcian blue (HID-AB) stain (pH 2.5) as
described previously [2]. For quantitative assessment of the mucin chemotypes, images of the HID-AB stained sections were captured on an Olympus EX51 Microscope (Olympus, Dublin, Ireland) and Cell^A image software (Olympus) at 209 magnification and subsequently analysed using the open source image analysis program IMAGEJ [17]. Colour-deconvolution was applied, thereby allowing the pixels belonging to each mucin type to be identified by the algorithm. For each section the area of sulphated and sialyated mucin was determined and results were expressed as the percentage of sulphated mucin relative to the total mucin content for a given section. For histopathological assessment of inflammation, 8lm sections were mounted onto plain glass slides and stained with Mayer’s haematoxylin followed by eosin counter staining (Sigma Aldrich, Dublin, Ireland) in a Leica Autostainer XL (Leica Microsystems, Ashbourne, Ireland) and subsequently mounted in DPX solution (Sigma Aldrich). All sections were scored by a pathologist (AM) according to a system previously described by Geboes et al. [18]. These scores were further categorized as no or mild inflammation (score 0–1), moderate inflammation (score 2.0–3.3) or severe inflammation (score 4.0–5.4). Laser capture microdissection and total DNA extraction
The ArcturusXTTM Laser Capture Microdissection System (Applied Biosystems, Paisley, UK) was used to isolate and laser capture colonic crypt material. LCM and subsequent microbiological analysis was limited to samples which displayed preserved architecture and an absence of overt crypt abscesses (i.e. a maximum inflammatory grade of 4.2).
FFPE & HID/AB staining Hisotological analysis of mucin chemotypes
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Figure 1 Diagrammatic representation of longitudinal and vertical sampling within the colon. Abbreviations: R, right colon; T, transverse colon; L, left colon; RE, rectum; FFPE, formlyn fixed paraffin embedded; HID/AB, high-iron diamine and Alcian blue; LCM, laser capture microdissection; qRT-PCR, quantitative real-time polymerase chain reaction.
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Briefly, 10-lm sections of snap-frozen biopsy were cut onto sterile, nuclease and nucleic acid free, PEN membrane slides (Applied Biosystems). Slides were stained briefly with filter-sterilized Alcian blue (AB) (Sigma Aldrich) and subsequently counterstained with Mayer’s haematoxylin (Sigma Aldrich) followed by dehydration with increasing concentrations (0, 25, 50, 75, 90 and 100%) of ethanol (Sigma Aldrich). Crypts were identified under 209 magnification and subsequently cryptassociated mucus was isolated from the basal membrane up to the MGL. LCM isolates were captured onto Arcturus CapSure LCM caps (Applied Biosystems), through both ultraviolet cutting and infrared spotting. The area of microdissected product was recorded for each sample. Following crypt isolation, total DNA was isolated with the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol for purification of total DNA from animal tissue. These extracts were subsequently stored at 20°C. Detection and quantification of pan-bacterial DNA within crypt extracts
The pan-bacterial DNA copy number within the crypt LCM products was determined through qRT-PCR. A primer and probe set previously described by Nadkarni et al. [19] were used for this purpose. Each qRT-PCR reaction was carried out in duplicate with multiple positive and negative controls included in each PCR assay run. All reactions were carried out in an ABI 7000 sequence detector (Applied Biosystems) using universal thermal cycling conditions: 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. Sequence detection software version 1.2 was used for all data analyses. A typical 20-ll real-time (RT) PCR amplification reaction contained 1 9 TaqMan Universal Mastermix (Applied Biosystems), the appropriate forward and reverse primers and a MGBNFQ probe at concentrations of 300 nM and 175 nM respectively, and 4 ll of DNA extract normalized to 1 ng/ll. Post-PCR analysis involved determination of the pan-bacterial copy number in each sample based on its fold change relative to a plasmid DNA standard. Subsequently, the calculated copy numbers were normalized for extract volume, concentration and microdissected area (mm2). Detection and quantification of DSV within crypt extracts
The 16S rRNA gene of DSV was detected and quantified as previously described [14]. RT-PCR reactions
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were carried out in duplicate wells on an ABI HT-7900 sequence detector (Applied Biosystems) and contained the following: 1 9 Taqman Universal master mix (Applied Biosystems), 100 nM of forward and reverse primers, 175 nM of probe and 4 ng of normalized DNA. The reactions were carried out under standard RT-PCR cycling condition outlined above. All assay runs were co-analysed with multiple positive and negative controls and included a set of DSV standards (ranging from 107 to 10 copies). A standard curve of threshold cycle and log [DSV plasmid] was generated. The DSV copy number per sample was generated from the standard curve and normalized for extracted volume, DNA concentration and microdissected area (mm2). All samples which returned a Ct result of < 40 were deemed to be positive for DSV. Construction of a plasmid DNA standard as a reference for determination of pan-bacterial and DSV copy number
A series of plasmid DNA standards was generated for the purpose of determining the pan-bacterial copy number and DSV copy number within each sample. These standards were included in each qRT-PCR assay run and used in a 2 DCt calculation to determine the fold difference in pan-bacterial copy number within each sample, and to generate a standard curve against which the DSV copy number could be determined. Briefly, Desulfovibrio desulfuricans (catalogue no. NCIMB12833; NCIMB Ltd, Aberdeen, UK) was cultured under anaerobic conditions in Postgate’s medium and DNA was extracted using a DNeasy Tissue Kit (Qiagen). The 466-bp amplicon generated by the primer set described above was amplified from the culture DNA extract and cloned into a pCR2.1-TOPO vector using the TOPO TA cloning system (Invitrogen, Groningen, The Netherlands) according to the manufacturer’s instructions. DNA from the recombinant plasmid mini-preps was purified using the QIAprep Spin Miniprep Kit (Qiagen). The total weight per recombinant plasmid was calculated and this was used to generate a series of DNA standards of known copy number of the target sequence. Statistical analysis
Mucin chemotype scores, haematoxylin and eosin scores, total bacterial copy number and patient demographics were used to generate a collective data set. The resulting file was inputted into the statistical analysis software program SPSS version 18 (Chicago, Illinois, USA) and nonparametric statistical analysis was carried out on all parameters. Descriptive statistics are presented
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in terms of median values and interquartile range (IQR) and graphed as box-plots on a logarithmic scale. Preliminary comparative analyses were performed by one-way ANOVA and Mann–Whitney U-testing based on the assumption that the data were compatible with ordinary linear models. Correlations were performed by Spearman rho correlations.
Results Mucin expression across the colon in health and UC
High-iron diamine and Alcian blue staining was used as a means of assessing the mucin chemotype within the colonic biopsy samples (Fig. 2a). Digital analysis of each biopsy section was performed to generate a quantitative measure of the mucin chemotype composition. Analysis revealed the presence of sulphated mucins in all biopsy samples from both cohorts and sulphomucin abundance to be greater in all HC biopsies with the exception of one individual. Within the AUC cohort sulphated
mucin remained the predominant mucin type; however, its abundance was reduced by < 50% in 12 biopsy samples from 7 individuals. Quantitative analysis revealed both regional and intercohort variation in the sulphomucin content of the biopsy samples. In health, the sulphated mucins were found to predominate throughout the colon, with a decreasing proximal to distal gradient in sulphation observed. Comparison of the four colonic regions revealed the right colon to have the highest abundance of sulphated mucin (median value 100%) and the rectum to have the lowest abundance (median value 85.60%) (Fig. 2b). A significant difference was observed between the right and left, and right and rectal regions but not between the other colonic regions (P < 0.05). Within the AUC cohort this decreasing mucin gradient was not apparent. A reduction in the presence of sulphated mucins was noted across the colon relative to that of the HC; however, no significant differences were observed between any of the colonic regions within the AUC cohort (Fig. 2b).
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Figure 2 Sulphomucin staining. (a) High-iron diamine and Alcian blue stained sections of human colonic tissue. Sulphomucins are stained brown/black and sialomucins are stained blue: (i) healthy control (HC), right colon 92.63% sulphation; (ii) acute ulcerative colitis (AUC), transverse colon 46.41% sulphation. (b) Intercohort comparison of the percentage of sulphation from HC and AUC across the longitudinal axis of the colon. HC are highlighted in green, AUC are highlighted in red. *,O Indicate outliers for each cohort.
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normalized DSV copy number was observed between cohorts (Fig. 3b,c).
A comparison of the sulphomucin abundance between the HC and AUC cohorts revealed a significant decrease in the presence of sulphomucin within the AUC cohort. When assessed as independent measures of each sampling region this decrease was significant for all four of the colonic regions examined (Fig. 2b).
Relationships between histological characterization, microbial colonization and patient demographics
A significant negative correlation between the abundance of sulphated mucin and inflammatory activity was observed, with a decrease in sulphated mucins corresponding to an increase in inflammation (P < 0.05). Furthermore, a weak but significant negative correlation was observed between the abundance of sulphated mucins and the normalized DSV copy numbers (R2 = 0.234, P < 0.05) (Fig. 4). No such correlation was noted for the total bacterial copy number. Likewise no significant correlation between the total bacterial, DSV, or normalized DSV copy number and inflammatory activity scores was noted. Neither relative DSV burden nor percentage sulphation was correlated with Mayo disease index scores.
Histological assessment of inflammatory activity across the colon in health and UC
Inflammation scores were categorized into mild, moderate and severe (mild, score 0–1; moderate, score 2.0– 3.3; severe, score 4.0–5.4). Significant differences were noted between both cohorts (P < 0.05). When examined on a regional basis, a significant increase in inflammation was noted at all regions of the colon in the AUC cohort relative to the HC. Total bacterial enumeration and DSV colonization in health and UC
A total bacterial signal was detected in all patients and at each region of the colon. The presence of DSV was found to be common but not ubiquitous. A total of six HC and two AUC individuals were not colonized with DSV. Total bacterial copy numbers were found to span over five orders of magnitude for the HC and four for the AUC cohort. No significant differences in the total bacterial copy number were found overall between cohorts or at each sampling region between the cohorts (Fig. 3a). Likewise for the quantification of DSV no significant difference in the DSV copy number or
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Discussion This study reports use of HID-AB staining in conjunction with image analysis to characterize quantitatively the histological changes in mucin chemotype expression both within goblet cells and in the expressed extracellular mucin in colonic biopsies obtained from patients with acutely active UC. These histological findings were compared with an analysis of the total bacterial and DSV colonization of the colonic crypts from parallel biopsy samples of each patient. The use of LCM in combination with qRT-PCR to enumerate
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Figure 3 Intercohort comparisons of: (a) total bacterial copy number per mm2 of laser capture microdissection (LCM) crypt material, (b) Desulfovibrio (DSV) copy number per mm2 of LCM crypt material, and (c) normalized DSV burden per mm2 of LCM crypt material. Individual healthy controls are highlighted in green and individuals with acute ulcerative colitis (UC) are highlighted in red. All results are plotted on a logarithmic scale. *,O Indicate outliers for each cohort.
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Figure 4 Normalized Desulfovibrio (DSV) burden per mm of laser capture microdissection crypt material compared with the percentage of sulphated mucins. The correlation coefficient is calculated by Spearman rho analysis. Individual healthy controls are highlighted in green and individuals with acute ulcerative colitis (UC) are highlighted in red.
the bacterial copy number present in the colonic crypts allowed a focused approach to studying the intact organization of the gastrointestinal microbiota directly within the crypt structures. The staining of cryosectioned material with AB prior to crypt isolation allowed the identification and isolation of not only intracellular mucins but also mucin which had been expressed into the crypt structures (Fig. 1). This is important as the colonic microbiota has been shown to conform to strict patterns of topographical distribution [20] and isolation of both intra- and extracellular mucins allows for a study of material within which bacteria reside. The presence of sulphomucins was identified in all biopsies from both cohorts analysed. Among the HCs, sulphated mucins were identified as the most abundant mucin chemotype in all colonic regions with the exception of a single biopsy. The observed decrease in sulphated mucins and corresponding increase in sialylated mucins in the proximal regions of the colon relative to the distal regions is consistent with previous reports [5,21]. Within the AUC cohort a change in mucin chemotype was noted with significant reduction in the presence of sulphated mucins and a corresponding increase in the presence of sialylated mucins. Additionally the decreasing proximal to distal gradient in
sulphated mucins observed within the HCs was absent from the AUC cohort. The reduction in sulphated mucin content is likely to reduce the protective function of the MGL and may place individuals at greater risk of damage from luminal substances due to reduced resistance of the MGL to bacterial degradation [8,9]. This observation was supported by the negative correlation found between the abundance of sulphated mucins and the degree of inflammation, suggesting that the colonic epithelium of individuals with decreased expression of sulphated mucin was more vulnerable to the toxic effects of luminal microbiota or their metabolome. In this context a bidirectional cause–effect relationship may exist between inflammation and deterioration of the MGL in UC. Alterations in mucin chemotype expression seen in the colon in UC may be related to the dysbiosis known to occur in the colitic colon [22]. In the present study, a combination of LCM and qRT-PCR provided a highly targeted approach to addressing this question based on sampling of crypt mucus without disrupting the integrity of the crypt. This approach has allowed examination of bacterial colonization rates directly at and within the crypt structures. Through normalization of our DSV results we have minimized potential reporting errors of bacterial copy numbers, so the normalized copy numbers reflect the true burden of DSV within the samples. No significant differences in the total bacterial, DSV or normalized DSV copy numbers were observed between cohorts in this study, a finding at odds with a previous observation from our group [14] and the hypothesis that the burden of DSV is greater in the colitic colon. However, in the present study, LCM and qRT-PCR analysis was restricted to crypts with preserved architecture, eliminating sites of overt crypt abscess. Thus the total bacterial copy numbers reported for this study are reflective of samples from areas of with inflammation but not severe ulceration and may explain the different findings in the present study. It is unlikely that the differences in sample collection and bowel preparation would have influenced the results of this study, as the mucus targeted during LCM was present within and adherent to the crypt structures and not on the luminal surface. It could also be argued that the method of sample preparation with regard to cryosectioning influenced the bacteriological results. It is now accepted that in health the MGL of the colon has a two-layered organization with an outer layer which is colonized by bacteria and an inner mucous layer devoid of bacteria [23]. Through sample preparation, surface bacteria could potentially contaminate the inner mucous layer; however, given that we chose to
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examine mucin which extends from the apical to the basal membrane of the crypt as a whole, the presence of bacteria in our samples is not surprising and any translocation of bacteria is negated by the design set up. It is also unlikely that relocation of bacteria from other areas of the sample has occurred as it has been reported that the adherence of bacteria to epithelial cells or their infiltration into the lamina propria is unlikely [21]. However, the present study did identify a potential link between normalized DSV burden (the most abundant SRB in the human colon [15]) and changes in mucin chemotype known to occur in UC. Decreasing sulphated mucin content was associated with increased abundance of DSV based on normalized gene copy numbers. The absence of this correlation with the absolute DSV copy numbers highlights the importance of normalizing species-specific results to the total bacterial load within a given sample. The negative correlation found between the colonic mucin chemotype and the commensal SRB present within the colon supports the hypothesis that a reduction in sulphated mucin chemotype expression characteristic of UC may in part relate to local abundance of SRB such as DSV within the colonic crypt niche. An alternative interpretation is that areas showing reduced sulphation are selectively more prone to colonization by DSV. Further appropriately powered studies combining clinical pathology and microbial gene expression are needed to clarify this and to ascertain the precise role or otherwise of DSV sulphate activity in the interrelationship between the microbiome and colonic mucin chemotype. This study has placed an emphasis on examining the presence of DSV; however, it should be considered that other species are capable of harvesting sulphate from colonic mucins through dissimilatory sulphate reduction. The Desulfobacter, Desulfobulbus and Desulfotomaculum bacteria are known commensals of the human colon and are also dependent on sulphate derived from sulphomucins for metabolism [9]. Potential syntrophic associations with mucolytic bacteria such as Akkermansia muciniphila, Ruminococcus gnavus and Ruminococcus totques also warrant consideration as mucin degradation and subsequent sulphate metabolism though hydrogenotrophy is the major metabolic pathway postulated for SRB in the colon [24,25]. In summary, this study provides evidence of an association between the presence of DSV and changes in mucin chemotype within the colitic colon. The reduction in abundance of sulphated mucin can be correlated to increased relative presence of DSV and increased inflammation suggesting these bacteria may play an important role in UC-associated inflammation.
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Author contributions PROC, JCC, NGD, DW, KS: conceived and designed experiments; GL, AB, NB, AL, AM: acquisition of data; GL, AB: analysis and interpretation of data; GL: wrote manuscript.
Funding Funding for this work was obtained through Science Foundation Ireland (GL, AB and AL) and the Bowel Disease Research Foundation (NB).
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20 Swidsinski A, Loening-Baucke V, Theissig F et al. Comparative study of the intestinal mucus barrier in normal and inflamed colon. Gut 2007; 56: 343–50. 21 Schultsz C, van den Berg FM, ten Kate FW, Tytgat GNJ, Dankert J. The intestinal mucus layer from patients with inflammatory bowel disease harbors high numbers of bacteria compared with controls. Gastroenterology 1999; 117: 1089–97. 22 Koropatkin NM, Cameron EA, Martens EC. How glycan metabolism shapes the human gut microbiota. Nat Rev Microbiol 2012; 10: 323–35. 23 Johansson MEV, Gustafsson JK, Holmen-Larsson J et al. Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis. Gut 2013; doi:10.1136/gutjnl2012-303207. 24 Png CW, Linden SK, Gilshenan KS et al. Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria. Am J Gastroenterol 2010; 105: 2420–8. 25 Derrien M, Vaughan EE, Plugge CM, de Vos WM. Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium. Int J Syst Evol Microbiol 2004; 54: 1469–76.
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Correlations between colonic crypt mucin chemotype, inflammatory grade and Desulfovibrio species in ulcerative colitis Balfe*†, N. Bambury*†, A. Lavelle*†, A. Maguire‡, N. G. Docherty§, G. Lennon*†, A. J. C. Coffey¶, D. C. Winter*†, K. Sheahan†‡ and P. R. O’Connell*† *School of Medicine and Medical Sciences, University College Dublin, Dublin, Ireland, †Centre for Colorectal Disease, St Vincent’s University Hospital, Dublin, Ireland, ‡Department of Histopathology, St Vincent’s University Hospital, Dublin, Ireland, §Department of Physiology, School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland and ¶Graduate Entry Medical School, University Hospital Limerick, University of Limerick, Limerick, Ireland Received 12 June 2013; accepted 2 October 2013; Accepted Article online 18 December 2013
Abstract Aim The colonic mucus gel layer is composed of mucins that may be sulphated or sialyated. Sulphated mucins predominate in health while in ulcerative colitis (UC) sulphation is reduced. These differences result directly from inflammatory events. It may also be hypothesized that they arise in part from alterations in the colonic microbiota, particularly changes in the burden of sulphated mucin-metabolizing species, such as Desulfovibrio (DSV) bacteria. The aim of this study was to correlate colonic mucin chemotypes and inflammatory scores in health and UC and relate these changes to changes in the colonization of colonic crypts by DSV. Method Paired colonic biopsies from 34 healthy controls (HC) and 19 patients with active UC were collected for the purpose of parallel histological and microbiological assessment. High-iron diamine and Alcian blue staining and haematoxylin and eosin of mucosal biopsy specimens were used to assess histological changes within the clinical spectrum of UC. Quantitative real-time polymerase chain reaction analysis was employed to determine the total and DSV copy number within the colonic crypts.
Results Compared with HC, the mucin chemotype in UC was less sulphated and inversely correlated with the degree of mucosal inflammation. A weak but significant negative correlation was found between the abundance of sulphated mucins and DSV burden. Conclusion Mucin composition strongly correlates with the degree of mucosal inflammation, and to a lesser extent with DSV burden. These data suggest that mucin chemotype and DSV burden are linked phenomena and highlight the need to consider changes in mucin chemotype in the setting of microbial dysbiosis occurring within the colitic colon. Keywords Ulcerative colitis, mucin chemotype, Desulfovibrio What does this paper add to the literature? Decreased sulphation of mucins has been associated with inflammation in ulcerative colitis. Currently there are few data describing the relationship between microbial species and changes in mucin chemotype. This study validates previous findings and presents evidence of changes in mucin chemotype occurring in tandem with coherent changes in the microbiota within crypt niches.
Introduction Correspondence to: Prof. P. Ronan O’Connell, Surgical Professorial Unit, St Vincent’s University Hospital, Elm Park, Dublin 4, Ireland E-mail: [email protected] Portions of this work were presented at the ESCP 2011 meeting in Copenhagen, Denmark and the SARS 2011 meeting in Dublin, Ireland and have been published in abstract form in Colorectal Disease 13 (Suppl 6), 2011 and British Journal of Surgery 99 (Suppl 4), 2012.
The mucus gel layer (MGL) in the colon is an important component of the innate immune system. It serves both as a barrier protecting colonic mucosa from harmful substances in the lumen and as an interface between the host epithelium and commensal bacteria present in the lumen [1]. In ulcerative colitis (UC) the MGL is quantitatively and qualitatively different from that in
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health, attributed to premature discharge of mucins from goblet cells [2,3]. The effects of changes in the glycoprotein and mucin content [4,5] on MGL function, and their relationship with the pathogenesis of UC, remain uncertain; however, both are likely to be important in the complex interplay between the colonic microbiota and epithelial immune responses. Mucin is a critical component of the MGL; it consists of a peptide backbone with alternating glycosylated and nonglycosylated domains [6]. Mucins can be classified as neutral or acidic, with the acidic mucins being further classified as sialyated or sulphated [7]. Sialyated mucins are converted to mature sulphated forms through posttranslational modification. Typically, the mucins expressed in the healthy human colon are predominantly sulphated [1,5]; however, in UC a predominance of unsulphated mucin (sialyated mucin type) has been observed [5,8,9] and the MGL is thinner due to a reduction in the volume of mucin present [3]. Type 2 mucin (MUC2) is the predominant mucin secreted by the colonic epithelium. A qualitative change in MUC2 structure occurs during acute inflammation, with an aberrant glycan profile occurring in patients with active UC [8,10]. These alterations in mucin type, together with a reduction in MGL thickness, may facilitate access of luminal bacteria or bacterial products to the underlying epithelium. Such changes are thought to be influenced by bacterial mucinases, including peptidases, glycosidases, sulphatases and esterases, all of which are increased in the colitic colon [9]. In health there is a constant equilibrium between synthesis of colonic mucins and breakdown by resident bacteria that possess the enzymes necessary to utilize colonic mucins as an energy source [11]. In health, colonic mucins are highly sulphated and this may act as a bacteriostatic factor providing resistance to enzymatic degradation [12]. Sulphation of mucins is also related to bacterial load, as evidenced by increased ratios of sulphated mucins in the mucosa of ileal pouch reservoirs, correlating with an increased luminal bacterial load [13]. Colonic crypts, which are laden with mucins, provide a niche for host–bacterial interaction [11,13]. Previously our group reported the use of laser capture microdissection (LCM) combined with quantitative real-time polymerase chain reaction (qRT-PCR) to study bacterial colonization of the colonic MGL. A decrease in total bacterial copy number and increase in Desulfovibrio (DSV) burden was noted at sites of severe inflammation and mucosal ulceration in specimens from patients with acute UC [13,14]. Desulfovibrio are common gut commensals and are the most abundant genus of sulphate-reducing bacteria (SRB) in the human colon [15]. These bacteria are
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capable of metabolizing colonic sulphomucin-derived sulphate, utilizing the sulphate for metabolism and in so doing producing hydrogen sulphide (H2S) gas which has potential genotoxic effects [12]. Whilst controversial, a body of evidence exists in the literature implicating SRB, DSV and H2S in UC-associated inflammation [12,16]. Therefore it could be hypothesized that the changes in mucin chemotype characteristic of UC may be driven not only by inflammation but also by changes in the abundance of SRB such as DSV. The aim of the present study was to quantify the expression of sulphated mucin chemotypes in health and acutely active UC and to investigate possible relationships between the mucin chemotype composition and the presence of DSV at the level of the preserved crypt structures within the colitic colon.
Method Patient recruitment
The experimental protocol was approved by the St Vincent’s University Hospital Ethics Committee, and written informed consent was obtained prior to sample collection. Enrolled subjects were over 18 years of age. Healthy controls (HC) had no history of inflammatory bowel disease (IBD) and were undergoing routine day case colonoscopy for polyp surveillance. Patients with acute UC (AUC) were recruited at the time of colectomy for chronic colitis refractory to medical treatment, acute severe colitis unresponsive to infliximab or fulminant colitis requiring emergency surgery. Subjects were excluded if they had received antibiotic treatment in the preceding 6 weeks, with the exception of patients with AUC who all received prophylactic broad-spectrum antibiotics at induction of anaesthesia. All HC had received bowel preparation with polyethylene glycol and sodium picosulphate prior to colonoscopy and sample collection. A total of 34 HC and 19 AUC patients were recruited to the study. The patient demographics for each cohort are given in Table 1. Sample collection
All samples were collected from HC subjects at the time of colonoscopy. Following successful caecal intubation, duplicate mucosal biopsies were taken at each of four levels (right colon, transverse colon, left colon and rectum) using a Radial Jaw3 biopsy forceps (Boston Scientific, Natick, Massachusetts, USA) (Fig. 1). Likewise four duplicate specimens were collected immediately following specimen delivery at the time of surgery in the AUC cohort. Biopsy specimens from both cohorts were
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Table 1 Summary of patient demographics in healthy controls and acute ulcerative colitis (UC).
Gender (male, female) Age (years) Smoking (n) Non-smoker Former smoker Current smoker Unknown Years since diagnosis Disease activity Mayo score Antibiotic treatment with previous 6 months*
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Acute UC
16, 18 55.5 [23–82]
9, 12 42.4 [23–83]
(12) (2) (3) (17) n/a
(6) (5) (0) (8) 8.5 [1–38]
n/a n/a
10 [8–11] (1)
Values given are mean [range], and (number of patients). *Refers to patients in receipt of antibiotic treatment 6 months prior undergoing the procedure but not within the 6-week exclusion period for this study.
processed in the same manner. One biopsy from each region sampled was placed in 10% neutral buffered formalin for a minimum of 24 h. Fixed biopsies were then paraffin embedded and used for histological analysis. The second biopsy specimen was placed in Tissue Tek OCT medium (Sakura Finetek Europe, Alphen aan den Rijn, The Netherlands), snap-frozen in liquid nitrogen and stored at 80°C for molecular analysis. Mucin histochemistry and histopathological analysis
To identify and differentiate between sulpho- and sialomucins, biopsy sections were stained with high-iron diamine and Alcian blue (HID-AB) stain (pH 2.5) as
described previously [2]. For quantitative assessment of the mucin chemotypes, images of the HID-AB stained sections were captured on an Olympus EX51 Microscope (Olympus, Dublin, Ireland) and Cell^A image software (Olympus) at 209 magnification and subsequently analysed using the open source image analysis program IMAGEJ [17]. Colour-deconvolution was applied, thereby allowing the pixels belonging to each mucin type to be identified by the algorithm. For each section the area of sulphated and sialyated mucin was determined and results were expressed as the percentage of sulphated mucin relative to the total mucin content for a given section. For histopathological assessment of inflammation, 8lm sections were mounted onto plain glass slides and stained with Mayer’s haematoxylin followed by eosin counter staining (Sigma Aldrich, Dublin, Ireland) in a Leica Autostainer XL (Leica Microsystems, Ashbourne, Ireland) and subsequently mounted in DPX solution (Sigma Aldrich). All sections were scored by a pathologist (AM) according to a system previously described by Geboes et al. [18]. These scores were further categorized as no or mild inflammation (score 0–1), moderate inflammation (score 2.0–3.3) or severe inflammation (score 4.0–5.4). Laser capture microdissection and total DNA extraction
The ArcturusXTTM Laser Capture Microdissection System (Applied Biosystems, Paisley, UK) was used to isolate and laser capture colonic crypt material. LCM and subsequent microbiological analysis was limited to samples which displayed preserved architecture and an absence of overt crypt abscesses (i.e. a maximum inflammatory grade of 4.2).
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Figure 1 Diagrammatic representation of longitudinal and vertical sampling within the colon. Abbreviations: R, right colon; T, transverse colon; L, left colon; RE, rectum; FFPE, formlyn fixed paraffin embedded; HID/AB, high-iron diamine and Alcian blue; LCM, laser capture microdissection; qRT-PCR, quantitative real-time polymerase chain reaction.
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Briefly, 10-lm sections of snap-frozen biopsy were cut onto sterile, nuclease and nucleic acid free, PEN membrane slides (Applied Biosystems). Slides were stained briefly with filter-sterilized Alcian blue (AB) (Sigma Aldrich) and subsequently counterstained with Mayer’s haematoxylin (Sigma Aldrich) followed by dehydration with increasing concentrations (0, 25, 50, 75, 90 and 100%) of ethanol (Sigma Aldrich). Crypts were identified under 209 magnification and subsequently cryptassociated mucus was isolated from the basal membrane up to the MGL. LCM isolates were captured onto Arcturus CapSure LCM caps (Applied Biosystems), through both ultraviolet cutting and infrared spotting. The area of microdissected product was recorded for each sample. Following crypt isolation, total DNA was isolated with the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol for purification of total DNA from animal tissue. These extracts were subsequently stored at 20°C. Detection and quantification of pan-bacterial DNA within crypt extracts
The pan-bacterial DNA copy number within the crypt LCM products was determined through qRT-PCR. A primer and probe set previously described by Nadkarni et al. [19] were used for this purpose. Each qRT-PCR reaction was carried out in duplicate with multiple positive and negative controls included in each PCR assay run. All reactions were carried out in an ABI 7000 sequence detector (Applied Biosystems) using universal thermal cycling conditions: 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. Sequence detection software version 1.2 was used for all data analyses. A typical 20-ll real-time (RT) PCR amplification reaction contained 1 9 TaqMan Universal Mastermix (Applied Biosystems), the appropriate forward and reverse primers and a MGBNFQ probe at concentrations of 300 nM and 175 nM respectively, and 4 ll of DNA extract normalized to 1 ng/ll. Post-PCR analysis involved determination of the pan-bacterial copy number in each sample based on its fold change relative to a plasmid DNA standard. Subsequently, the calculated copy numbers were normalized for extract volume, concentration and microdissected area (mm2). Detection and quantification of DSV within crypt extracts
The 16S rRNA gene of DSV was detected and quantified as previously described [14]. RT-PCR reactions
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were carried out in duplicate wells on an ABI HT-7900 sequence detector (Applied Biosystems) and contained the following: 1 9 Taqman Universal master mix (Applied Biosystems), 100 nM of forward and reverse primers, 175 nM of probe and 4 ng of normalized DNA. The reactions were carried out under standard RT-PCR cycling condition outlined above. All assay runs were co-analysed with multiple positive and negative controls and included a set of DSV standards (ranging from 107 to 10 copies). A standard curve of threshold cycle and log [DSV plasmid] was generated. The DSV copy number per sample was generated from the standard curve and normalized for extracted volume, DNA concentration and microdissected area (mm2). All samples which returned a Ct result of < 40 were deemed to be positive for DSV. Construction of a plasmid DNA standard as a reference for determination of pan-bacterial and DSV copy number
A series of plasmid DNA standards was generated for the purpose of determining the pan-bacterial copy number and DSV copy number within each sample. These standards were included in each qRT-PCR assay run and used in a 2 DCt calculation to determine the fold difference in pan-bacterial copy number within each sample, and to generate a standard curve against which the DSV copy number could be determined. Briefly, Desulfovibrio desulfuricans (catalogue no. NCIMB12833; NCIMB Ltd, Aberdeen, UK) was cultured under anaerobic conditions in Postgate’s medium and DNA was extracted using a DNeasy Tissue Kit (Qiagen). The 466-bp amplicon generated by the primer set described above was amplified from the culture DNA extract and cloned into a pCR2.1-TOPO vector using the TOPO TA cloning system (Invitrogen, Groningen, The Netherlands) according to the manufacturer’s instructions. DNA from the recombinant plasmid mini-preps was purified using the QIAprep Spin Miniprep Kit (Qiagen). The total weight per recombinant plasmid was calculated and this was used to generate a series of DNA standards of known copy number of the target sequence. Statistical analysis
Mucin chemotype scores, haematoxylin and eosin scores, total bacterial copy number and patient demographics were used to generate a collective data set. The resulting file was inputted into the statistical analysis software program SPSS version 18 (Chicago, Illinois, USA) and nonparametric statistical analysis was carried out on all parameters. Descriptive statistics are presented
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in terms of median values and interquartile range (IQR) and graphed as box-plots on a logarithmic scale. Preliminary comparative analyses were performed by one-way ANOVA and Mann–Whitney U-testing based on the assumption that the data were compatible with ordinary linear models. Correlations were performed by Spearman rho correlations.
Results Mucin expression across the colon in health and UC
High-iron diamine and Alcian blue staining was used as a means of assessing the mucin chemotype within the colonic biopsy samples (Fig. 2a). Digital analysis of each biopsy section was performed to generate a quantitative measure of the mucin chemotype composition. Analysis revealed the presence of sulphated mucins in all biopsy samples from both cohorts and sulphomucin abundance to be greater in all HC biopsies with the exception of one individual. Within the AUC cohort sulphated
mucin remained the predominant mucin type; however, its abundance was reduced by < 50% in 12 biopsy samples from 7 individuals. Quantitative analysis revealed both regional and intercohort variation in the sulphomucin content of the biopsy samples. In health, the sulphated mucins were found to predominate throughout the colon, with a decreasing proximal to distal gradient in sulphation observed. Comparison of the four colonic regions revealed the right colon to have the highest abundance of sulphated mucin (median value 100%) and the rectum to have the lowest abundance (median value 85.60%) (Fig. 2b). A significant difference was observed between the right and left, and right and rectal regions but not between the other colonic regions (P < 0.05). Within the AUC cohort this decreasing mucin gradient was not apparent. A reduction in the presence of sulphated mucins was noted across the colon relative to that of the HC; however, no significant differences were observed between any of the colonic regions within the AUC cohort (Fig. 2b).
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Figure 2 Sulphomucin staining. (a) High-iron diamine and Alcian blue stained sections of human colonic tissue. Sulphomucins are stained brown/black and sialomucins are stained blue: (i) healthy control (HC), right colon 92.63% sulphation; (ii) acute ulcerative colitis (AUC), transverse colon 46.41% sulphation. (b) Intercohort comparison of the percentage of sulphation from HC and AUC across the longitudinal axis of the colon. HC are highlighted in green, AUC are highlighted in red. *,O Indicate outliers for each cohort.
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normalized DSV copy number was observed between cohorts (Fig. 3b,c).
A comparison of the sulphomucin abundance between the HC and AUC cohorts revealed a significant decrease in the presence of sulphomucin within the AUC cohort. When assessed as independent measures of each sampling region this decrease was significant for all four of the colonic regions examined (Fig. 2b).
Relationships between histological characterization, microbial colonization and patient demographics
A significant negative correlation between the abundance of sulphated mucin and inflammatory activity was observed, with a decrease in sulphated mucins corresponding to an increase in inflammation (P < 0.05). Furthermore, a weak but significant negative correlation was observed between the abundance of sulphated mucins and the normalized DSV copy numbers (R2 = 0.234, P < 0.05) (Fig. 4). No such correlation was noted for the total bacterial copy number. Likewise no significant correlation between the total bacterial, DSV, or normalized DSV copy number and inflammatory activity scores was noted. Neither relative DSV burden nor percentage sulphation was correlated with Mayo disease index scores.
Histological assessment of inflammatory activity across the colon in health and UC
Inflammation scores were categorized into mild, moderate and severe (mild, score 0–1; moderate, score 2.0– 3.3; severe, score 4.0–5.4). Significant differences were noted between both cohorts (P < 0.05). When examined on a regional basis, a significant increase in inflammation was noted at all regions of the colon in the AUC cohort relative to the HC. Total bacterial enumeration and DSV colonization in health and UC
A total bacterial signal was detected in all patients and at each region of the colon. The presence of DSV was found to be common but not ubiquitous. A total of six HC and two AUC individuals were not colonized with DSV. Total bacterial copy numbers were found to span over five orders of magnitude for the HC and four for the AUC cohort. No significant differences in the total bacterial copy number were found overall between cohorts or at each sampling region between the cohorts (Fig. 3a). Likewise for the quantification of DSV no significant difference in the DSV copy number or
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Discussion This study reports use of HID-AB staining in conjunction with image analysis to characterize quantitatively the histological changes in mucin chemotype expression both within goblet cells and in the expressed extracellular mucin in colonic biopsies obtained from patients with acutely active UC. These histological findings were compared with an analysis of the total bacterial and DSV colonization of the colonic crypts from parallel biopsy samples of each patient. The use of LCM in combination with qRT-PCR to enumerate
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Figure 3 Intercohort comparisons of: (a) total bacterial copy number per mm2 of laser capture microdissection (LCM) crypt material, (b) Desulfovibrio (DSV) copy number per mm2 of LCM crypt material, and (c) normalized DSV burden per mm2 of LCM crypt material. Individual healthy controls are highlighted in green and individuals with acute ulcerative colitis (UC) are highlighted in red. All results are plotted on a logarithmic scale. *,O Indicate outliers for each cohort.
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Figure 4 Normalized Desulfovibrio (DSV) burden per mm of laser capture microdissection crypt material compared with the percentage of sulphated mucins. The correlation coefficient is calculated by Spearman rho analysis. Individual healthy controls are highlighted in green and individuals with acute ulcerative colitis (UC) are highlighted in red.
the bacterial copy number present in the colonic crypts allowed a focused approach to studying the intact organization of the gastrointestinal microbiota directly within the crypt structures. The staining of cryosectioned material with AB prior to crypt isolation allowed the identification and isolation of not only intracellular mucins but also mucin which had been expressed into the crypt structures (Fig. 1). This is important as the colonic microbiota has been shown to conform to strict patterns of topographical distribution [20] and isolation of both intra- and extracellular mucins allows for a study of material within which bacteria reside. The presence of sulphomucins was identified in all biopsies from both cohorts analysed. Among the HCs, sulphated mucins were identified as the most abundant mucin chemotype in all colonic regions with the exception of a single biopsy. The observed decrease in sulphated mucins and corresponding increase in sialylated mucins in the proximal regions of the colon relative to the distal regions is consistent with previous reports [5,21]. Within the AUC cohort a change in mucin chemotype was noted with significant reduction in the presence of sulphated mucins and a corresponding increase in the presence of sialylated mucins. Additionally the decreasing proximal to distal gradient in
sulphated mucins observed within the HCs was absent from the AUC cohort. The reduction in sulphated mucin content is likely to reduce the protective function of the MGL and may place individuals at greater risk of damage from luminal substances due to reduced resistance of the MGL to bacterial degradation [8,9]. This observation was supported by the negative correlation found between the abundance of sulphated mucins and the degree of inflammation, suggesting that the colonic epithelium of individuals with decreased expression of sulphated mucin was more vulnerable to the toxic effects of luminal microbiota or their metabolome. In this context a bidirectional cause–effect relationship may exist between inflammation and deterioration of the MGL in UC. Alterations in mucin chemotype expression seen in the colon in UC may be related to the dysbiosis known to occur in the colitic colon [22]. In the present study, a combination of LCM and qRT-PCR provided a highly targeted approach to addressing this question based on sampling of crypt mucus without disrupting the integrity of the crypt. This approach has allowed examination of bacterial colonization rates directly at and within the crypt structures. Through normalization of our DSV results we have minimized potential reporting errors of bacterial copy numbers, so the normalized copy numbers reflect the true burden of DSV within the samples. No significant differences in the total bacterial, DSV or normalized DSV copy numbers were observed between cohorts in this study, a finding at odds with a previous observation from our group [14] and the hypothesis that the burden of DSV is greater in the colitic colon. However, in the present study, LCM and qRT-PCR analysis was restricted to crypts with preserved architecture, eliminating sites of overt crypt abscess. Thus the total bacterial copy numbers reported for this study are reflective of samples from areas of with inflammation but not severe ulceration and may explain the different findings in the present study. It is unlikely that the differences in sample collection and bowel preparation would have influenced the results of this study, as the mucus targeted during LCM was present within and adherent to the crypt structures and not on the luminal surface. It could also be argued that the method of sample preparation with regard to cryosectioning influenced the bacteriological results. It is now accepted that in health the MGL of the colon has a two-layered organization with an outer layer which is colonized by bacteria and an inner mucous layer devoid of bacteria [23]. Through sample preparation, surface bacteria could potentially contaminate the inner mucous layer; however, given that we chose to
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examine mucin which extends from the apical to the basal membrane of the crypt as a whole, the presence of bacteria in our samples is not surprising and any translocation of bacteria is negated by the design set up. It is also unlikely that relocation of bacteria from other areas of the sample has occurred as it has been reported that the adherence of bacteria to epithelial cells or their infiltration into the lamina propria is unlikely [21]. However, the present study did identify a potential link between normalized DSV burden (the most abundant SRB in the human colon [15]) and changes in mucin chemotype known to occur in UC. Decreasing sulphated mucin content was associated with increased abundance of DSV based on normalized gene copy numbers. The absence of this correlation with the absolute DSV copy numbers highlights the importance of normalizing species-specific results to the total bacterial load within a given sample. The negative correlation found between the colonic mucin chemotype and the commensal SRB present within the colon supports the hypothesis that a reduction in sulphated mucin chemotype expression characteristic of UC may in part relate to local abundance of SRB such as DSV within the colonic crypt niche. An alternative interpretation is that areas showing reduced sulphation are selectively more prone to colonization by DSV. Further appropriately powered studies combining clinical pathology and microbial gene expression are needed to clarify this and to ascertain the precise role or otherwise of DSV sulphate activity in the interrelationship between the microbiome and colonic mucin chemotype. This study has placed an emphasis on examining the presence of DSV; however, it should be considered that other species are capable of harvesting sulphate from colonic mucins through dissimilatory sulphate reduction. The Desulfobacter, Desulfobulbus and Desulfotomaculum bacteria are known commensals of the human colon and are also dependent on sulphate derived from sulphomucins for metabolism [9]. Potential syntrophic associations with mucolytic bacteria such as Akkermansia muciniphila, Ruminococcus gnavus and Ruminococcus totques also warrant consideration as mucin degradation and subsequent sulphate metabolism though hydrogenotrophy is the major metabolic pathway postulated for SRB in the colon [24,25]. In summary, this study provides evidence of an association between the presence of DSV and changes in mucin chemotype within the colitic colon. The reduction in abundance of sulphated mucin can be correlated to increased relative presence of DSV and increased inflammation suggesting these bacteria may play an important role in UC-associated inflammation.
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Author contributions PROC, JCC, NGD, DW, KS: conceived and designed experiments; GL, AB, NB, AL, AM: acquisition of data; GL, AB: analysis and interpretation of data; GL: wrote manuscript.
Funding Funding for this work was obtained through Science Foundation Ireland (GL, AB and AL) and the Bowel Disease Research Foundation (NB).
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