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Stereomicroscopic 3D-pattern profiling of murine and human intestinal inflammation reveals unique structural phenotypes.

Rodriguez-Palacios A, Kodani T, Kaydo L, Pietropaoli D, Corridoni D, Howell S, Katz J, Xin W, Pizarro TT, Cominelli F - Nat Commun (2015)

Bottom Line: We examine the mucosal surface of >700 mice (encompassing >16 strains and various IBD-models), create a profiling catalogue of 3D-stereomicroscopic abnormalities and demonstrate that mice with comparable histological scores display unique sub-clusters of 3D-structure-patterns of IBD pathology, which we call 3D-stereoenterotypes, and which are otherwise indiscernible histologically.We show that two ileal IBD-stereoenterotypes ('cobblestones' versus 'villous mini-aggregation') cluster separately within two distinct mouse lines of spontaneous ileitis, suggesting that host genetics drive unique and divergent inflammatory 3D-structural patterns in the gut.We suggest that stereomicroscopic (3D-SMAPgut) profiling can be easily implemented and enable the comprehensive study of inflammatory 3D structures, genetics and flora in IBD.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology and Liver Disease, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.

ABSTRACT
Histology is fundamental to assess two-dimensional intestinal inflammation; however, inflammatory bowel diseases (IBDs) are often indistinguishable microscopically on the basis of mucosal biopsies. Here, we use stereomicroscopy (SM) to rapidly profile the entire intestinal topography and assess inflammation. We examine the mucosal surface of >700 mice (encompassing >16 strains and various IBD-models), create a profiling catalogue of 3D-stereomicroscopic abnormalities and demonstrate that mice with comparable histological scores display unique sub-clusters of 3D-structure-patterns of IBD pathology, which we call 3D-stereoenterotypes, and which are otherwise indiscernible histologically. We show that two ileal IBD-stereoenterotypes ('cobblestones' versus 'villous mini-aggregation') cluster separately within two distinct mouse lines of spontaneous ileitis, suggesting that host genetics drive unique and divergent inflammatory 3D-structural patterns in the gut. In humans, stereomicroscopy reveals 'liquefaction' lesions and hierarchical fistulous complexes, enriched with clostridia/segmented filamentous bacteria, running under healthy mucosa in Crohn's disease. We suggest that stereomicroscopic (3D-SMAPgut) profiling can be easily implemented and enable the comprehensive study of inflammatory 3D structures, genetics and flora in IBD.

No MeSH data available.


Related in: MedlinePlus

Re-scaling of MPO data for weighted-3D-SAMPgut and gene expression analyses in mice.(a) MPO data from Fig. 2f is not normally distributed (P>0.1) and therefore not comparable for weighted analysis across SM-dissected tissues. Log-transformation allowed comparable normalization for ileum and colon (n=150 samples; B6, AKR, SAMP). (b) Re-scaling of MPO activity for adjustment of SM scores (percentage of abnormal mucosa). Log-transformation favours differentiation of samples with low MPO (steepest, below curve shoulder ∼200 U g−1). (c) Raw qPCR-CT gene expression values correlation plots versus re-scaled MPO activity. In β-actin panel (narrowest variability), y=0.603x represents linear correlation slope (for each unit of increase in re-scaled-MPO, β-actin increased by 0.603 units); value 15.2 indicates intercept. Low R2 indicates variability departing from fitted line cannot be explained by equation or MPO activity alone. (d) Example of SM-targeted 2−ddCT-fold analysis of il6 and tlr4 genes (six samples per mouse) for two randomly selected dexamethasone-treated mice. Note reproducible regional expression profile variability within colon (tlr4:il6 ratio is highest distally, but lowest proximally), suggesting SM-guided studies are best by normal–abnormal paired sampling within the same region. Paired SM sampling of ileum shows also a pattern: high tlr4:il6 ratio in normal areas, but low in cobblestones. Paired sampling may reveal biologically relevant spatial/structural differences within the same mouse strain: for example, il6 is overexpressed in proximal colon, which is less inflamed in DSS-colitis, but also in small intestinal cobblestones which are markedly inflamed in SAMP ileitis. IL6 varies (anti/proinflammatory) in the same mouse strain depending on location and SM-lesion structure.
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f3: Re-scaling of MPO data for weighted-3D-SAMPgut and gene expression analyses in mice.(a) MPO data from Fig. 2f is not normally distributed (P>0.1) and therefore not comparable for weighted analysis across SM-dissected tissues. Log-transformation allowed comparable normalization for ileum and colon (n=150 samples; B6, AKR, SAMP). (b) Re-scaling of MPO activity for adjustment of SM scores (percentage of abnormal mucosa). Log-transformation favours differentiation of samples with low MPO (steepest, below curve shoulder ∼200 U g−1). (c) Raw qPCR-CT gene expression values correlation plots versus re-scaled MPO activity. In β-actin panel (narrowest variability), y=0.603x represents linear correlation slope (for each unit of increase in re-scaled-MPO, β-actin increased by 0.603 units); value 15.2 indicates intercept. Low R2 indicates variability departing from fitted line cannot be explained by equation or MPO activity alone. (d) Example of SM-targeted 2−ddCT-fold analysis of il6 and tlr4 genes (six samples per mouse) for two randomly selected dexamethasone-treated mice. Note reproducible regional expression profile variability within colon (tlr4:il6 ratio is highest distally, but lowest proximally), suggesting SM-guided studies are best by normal–abnormal paired sampling within the same region. Paired SM sampling of ileum shows also a pattern: high tlr4:il6 ratio in normal areas, but low in cobblestones. Paired sampling may reveal biologically relevant spatial/structural differences within the same mouse strain: for example, il6 is overexpressed in proximal colon, which is less inflamed in DSS-colitis, but also in small intestinal cobblestones which are markedly inflamed in SAMP ileitis. IL6 varies (anti/proinflammatory) in the same mouse strain depending on location and SM-lesion structure.

Mentions: Because the percentage of 3D abnormal mucosa may not change (heal) with some treatments, but inflammation could decrease transmurally, we established that, if desired, the percentage of abnormal mucosa could be mathematically adjusted with its MPO value to create a biochemistry-weighted 3D-SMAP system to differentiate intestinal responses using a biochemical marker. This approach is useful if two mice had the same percentage of abnormal mucosa. We discovered that differentiation is possible by multiplying the percentage of abnormal (and normal) mucosa by their respective MPO values after re-scaling the Log2-normalized MPO data (Fig. 3a,b). Re-scaled MPO (ranging between 1 and 2, minimum and maximum for given laboratory/experiment, see Methods) doubles the SM percentage value when MPOs are highest (value 2), or has no impact when MPOs are lowest (value 1). Although the addition of the MPO-weighted SM percentage areas is simple and provides a comprehensive linearized measurement of disease extent and inflammation severity (in arbitrary scale, 0–200), analysis of data is also possible separately using multivariate statistics. The MPO-weighted SM concept may apply to other biochemical markers, and it is a simplified integrated multivariable attempt to aid researchers with intestinal phenotyping.


Stereomicroscopic 3D-pattern profiling of murine and human intestinal inflammation reveals unique structural phenotypes.

Rodriguez-Palacios A, Kodani T, Kaydo L, Pietropaoli D, Corridoni D, Howell S, Katz J, Xin W, Pizarro TT, Cominelli F - Nat Commun (2015)

Re-scaling of MPO data for weighted-3D-SAMPgut and gene expression analyses in mice.(a) MPO data from Fig. 2f is not normally distributed (P>0.1) and therefore not comparable for weighted analysis across SM-dissected tissues. Log-transformation allowed comparable normalization for ileum and colon (n=150 samples; B6, AKR, SAMP). (b) Re-scaling of MPO activity for adjustment of SM scores (percentage of abnormal mucosa). Log-transformation favours differentiation of samples with low MPO (steepest, below curve shoulder ∼200 U g−1). (c) Raw qPCR-CT gene expression values correlation plots versus re-scaled MPO activity. In β-actin panel (narrowest variability), y=0.603x represents linear correlation slope (for each unit of increase in re-scaled-MPO, β-actin increased by 0.603 units); value 15.2 indicates intercept. Low R2 indicates variability departing from fitted line cannot be explained by equation or MPO activity alone. (d) Example of SM-targeted 2−ddCT-fold analysis of il6 and tlr4 genes (six samples per mouse) for two randomly selected dexamethasone-treated mice. Note reproducible regional expression profile variability within colon (tlr4:il6 ratio is highest distally, but lowest proximally), suggesting SM-guided studies are best by normal–abnormal paired sampling within the same region. Paired SM sampling of ileum shows also a pattern: high tlr4:il6 ratio in normal areas, but low in cobblestones. Paired sampling may reveal biologically relevant spatial/structural differences within the same mouse strain: for example, il6 is overexpressed in proximal colon, which is less inflamed in DSS-colitis, but also in small intestinal cobblestones which are markedly inflamed in SAMP ileitis. IL6 varies (anti/proinflammatory) in the same mouse strain depending on location and SM-lesion structure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4510646&req=5

f3: Re-scaling of MPO data for weighted-3D-SAMPgut and gene expression analyses in mice.(a) MPO data from Fig. 2f is not normally distributed (P>0.1) and therefore not comparable for weighted analysis across SM-dissected tissues. Log-transformation allowed comparable normalization for ileum and colon (n=150 samples; B6, AKR, SAMP). (b) Re-scaling of MPO activity for adjustment of SM scores (percentage of abnormal mucosa). Log-transformation favours differentiation of samples with low MPO (steepest, below curve shoulder ∼200 U g−1). (c) Raw qPCR-CT gene expression values correlation plots versus re-scaled MPO activity. In β-actin panel (narrowest variability), y=0.603x represents linear correlation slope (for each unit of increase in re-scaled-MPO, β-actin increased by 0.603 units); value 15.2 indicates intercept. Low R2 indicates variability departing from fitted line cannot be explained by equation or MPO activity alone. (d) Example of SM-targeted 2−ddCT-fold analysis of il6 and tlr4 genes (six samples per mouse) for two randomly selected dexamethasone-treated mice. Note reproducible regional expression profile variability within colon (tlr4:il6 ratio is highest distally, but lowest proximally), suggesting SM-guided studies are best by normal–abnormal paired sampling within the same region. Paired SM sampling of ileum shows also a pattern: high tlr4:il6 ratio in normal areas, but low in cobblestones. Paired sampling may reveal biologically relevant spatial/structural differences within the same mouse strain: for example, il6 is overexpressed in proximal colon, which is less inflamed in DSS-colitis, but also in small intestinal cobblestones which are markedly inflamed in SAMP ileitis. IL6 varies (anti/proinflammatory) in the same mouse strain depending on location and SM-lesion structure.
Mentions: Because the percentage of 3D abnormal mucosa may not change (heal) with some treatments, but inflammation could decrease transmurally, we established that, if desired, the percentage of abnormal mucosa could be mathematically adjusted with its MPO value to create a biochemistry-weighted 3D-SMAP system to differentiate intestinal responses using a biochemical marker. This approach is useful if two mice had the same percentage of abnormal mucosa. We discovered that differentiation is possible by multiplying the percentage of abnormal (and normal) mucosa by their respective MPO values after re-scaling the Log2-normalized MPO data (Fig. 3a,b). Re-scaled MPO (ranging between 1 and 2, minimum and maximum for given laboratory/experiment, see Methods) doubles the SM percentage value when MPOs are highest (value 2), or has no impact when MPOs are lowest (value 1). Although the addition of the MPO-weighted SM percentage areas is simple and provides a comprehensive linearized measurement of disease extent and inflammation severity (in arbitrary scale, 0–200), analysis of data is also possible separately using multivariate statistics. The MPO-weighted SM concept may apply to other biochemical markers, and it is a simplified integrated multivariable attempt to aid researchers with intestinal phenotyping.

Bottom Line: We examine the mucosal surface of >700 mice (encompassing >16 strains and various IBD-models), create a profiling catalogue of 3D-stereomicroscopic abnormalities and demonstrate that mice with comparable histological scores display unique sub-clusters of 3D-structure-patterns of IBD pathology, which we call 3D-stereoenterotypes, and which are otherwise indiscernible histologically.We show that two ileal IBD-stereoenterotypes ('cobblestones' versus 'villous mini-aggregation') cluster separately within two distinct mouse lines of spontaneous ileitis, suggesting that host genetics drive unique and divergent inflammatory 3D-structural patterns in the gut.We suggest that stereomicroscopic (3D-SMAPgut) profiling can be easily implemented and enable the comprehensive study of inflammatory 3D structures, genetics and flora in IBD.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology and Liver Disease, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.

ABSTRACT
Histology is fundamental to assess two-dimensional intestinal inflammation; however, inflammatory bowel diseases (IBDs) are often indistinguishable microscopically on the basis of mucosal biopsies. Here, we use stereomicroscopy (SM) to rapidly profile the entire intestinal topography and assess inflammation. We examine the mucosal surface of >700 mice (encompassing >16 strains and various IBD-models), create a profiling catalogue of 3D-stereomicroscopic abnormalities and demonstrate that mice with comparable histological scores display unique sub-clusters of 3D-structure-patterns of IBD pathology, which we call 3D-stereoenterotypes, and which are otherwise indiscernible histologically. We show that two ileal IBD-stereoenterotypes ('cobblestones' versus 'villous mini-aggregation') cluster separately within two distinct mouse lines of spontaneous ileitis, suggesting that host genetics drive unique and divergent inflammatory 3D-structural patterns in the gut. In humans, stereomicroscopy reveals 'liquefaction' lesions and hierarchical fistulous complexes, enriched with clostridia/segmented filamentous bacteria, running under healthy mucosa in Crohn's disease. We suggest that stereomicroscopic (3D-SMAPgut) profiling can be easily implemented and enable the comprehensive study of inflammatory 3D structures, genetics and flora in IBD.

No MeSH data available.


Related in: MedlinePlus