Ischemia-induced mucus barrier loss and bacterial penetration are rapidly
counteracted by increased goblet cell secretory activity in human and rat colon
J Grootjans1, IH Hundscheid1, K Lenaerts1, B Boonen1, IB Renes2, FK Verheyen3,
CH Dejong1, MF von Meyenfeldt1, GL Beets1, WA Buurman1
1
Department of Surgery, NUTRIM School for Nutrition, Toxicology & Metabolism,
Maastricht University Medical Center, Maastricht, the Netherlands
2
Laboratory of Pediatrics, Division of Neonatology, Erasmus MC-Sophia, Rotterdam,
the Netherlands
3
Department of Molecular Cell Biology, Electron Microscopy Unit, Maastricht
University Medical Center, Maastricht, the Netherlands
Ischemia/reperfusion (IR) of the colon is a frequently observed event in clinical
practice carrying high morbidity. The high morbidity is associated with intestinal
barrier function loss, leading to bacterial translocation and severe inflammation. In
the colon, an important first line of defense against intruding pathogens is the mucus
layer, which is produced by goblet cells. In this study, we investigated consequences
of colon IR on the mucus layer and goblet cells, in a newly developed human and rat
experimental IR model.
In 10 patients, a small part of colon that had to be removed for surgical reasons was
isolated and exposed to 60 minutes of ischemia with 0, 30 or 60 minutes of
reperfusion (60I, 30R and 60R, respectively). Tissue not exposed to IR served as
control. In rats, colon was exposed to 60I, 60I30R, 60I120R or 60I 240R (n=7 per
group). Human and rat tissue was either snap-frozen, fixed in glutaraldehyde for
electron microscopy (EM), fixed in formalin or fixed in methacarn fixative to preserve
the mucus layer. qPCR was performed for MUC2, IL-6, IL1- and TNF-. The mucus
layer
and
goblet
cells
were
assessed
using
PAS/Alcian
Blue
(AB)
or
immunohistochemistry for MUC2/Dolichos biflorus agglutinin (DBA). Bacteria were
studied using EM and fluorescent in situ hybridization (FISH) for bacterial rRNA.
Neutrophil influx was studied using Myeloperoxidase staining.
PAS/AB and MUC2/DBA staining revealed breaches in the mucus layer at 60
minutes of ischemia. This was accompanied by penetration of bacteria into the
normally sterile crypts, as observed in EM and FISH, which induced massive release
of goblet cell granules. During reperfusion, increased goblet cell secretory activity led
to expulsion of bacteria from the bottom of the crypts towards the crypt surface. At
240 minutes of reperfusion, no bacteria were observed near the epithelium and a
newly formed mucus layer provided spatial separation of intraluminal bacteria with
the epithelial lining. Inflammation was limited to a slight influx of neutrophils into the
lamina propria and increased expression of IL-6, IL1- and TNF-.
In conclusion, this study provides first clues for the pathophysiology of human colonic
IR. Colon ischemia is associated with breaches in the mucus layer and bacterial
adherence to the epithelium. This is rapidly counteracted by increased secretory
activity of goblet cells, leading to expulsion of bacteria from the crypts as well as
restoration of the mucus barrier. This unique mechanism of the colon limits IRinduced bacterial penetration and inflammation.