Restitution of scratch-wounded IEC-18 monolayers is marked by migration of adjacent cells.

<p>A: representative sequence of images taken every hour (from top to bottom) beginning directly after mechanical wounding of a confluent IEC-18 monolayer. The approximate location of the initial wound margin (top picture) is indicated by a green dashed line and also displayed in subsequent images. Scale bar: 100μm. B: Representative images of migrating cells sending ahead filopodia (red arrows) and pseudopodia (orange arrows). C: Exemplary sequence of processes at the wound margin. Two adjacent cells are edged in red and shown at the indicated time points. Over time they migrate into the denuded area undergoing considerable morphological changes. D: Exemplary sequence of events within the cell sheet distant from the wound. Two representative cells are edged in red and show only bare movements or changes in shape over six hours.</p

Boyden chamber results.

<p>A: Representative images of stained cells attached to the lower surface of polycarbonate membranes after migration of IEC-18 towards medium containing Clt or not and after or without IFN-γ pretreatment. B: Quantitative analysis of the number of cells migrated through a polycarbonate membrane during six hours under the indicated conditions (n = 14–16). Clt-dependent migration is significantly higher under baseline than under inflammatory conditions.</p

Hypothesized impact of KCNN4 inhibition on wound-induced migration and differential regulation by IFN-γ.

<p>A: Blockade of KCNN4 by Clt (red X) accelerates wound healing at least in part by transactivation of the PI3K pathway (red arrows). Additional EGF (blue) meets activated downstream signaling and exerts no additional effect (blue arrows). B: IFN-γ preincubation uncouples the postulated transactivation of PI3K signaling by KCNN4 inhibition (orange line) leading to reduced migration upon KCNN4 blockade by Clt (red X). In this constellation EGF (blue) may activate downstream targets and enhance migration (blue arrows).</p

Clt-dependent alterations in Akt but not ERK phosphorylation correlate with wound healing of IEC-18.

<p>A: Representative blots showing Akt phosphorylation after 30 and 120 minutes. B: Representative blots showing ERK phosphorylation after 30 and 120 minutes. n.w.–not wounded. C-E: Quantitative analysis of the phosphorylation of Akt (left panels) and ERK (right panels) as determined by pAkt/Akt and pERK/ERK, respectively, after 30 (C), 120 (D) and 480 minutes (E). Clt enhances Akt but not ERK phosphorylation after 30 and 120 minutes. n = 3–7. F: Phosphorylation of Akt (upper panel) and ERK (lower panel) in Clt-involving conditions 120 minutes after wounding (n = 3–5). While ERK phosphorylation is not significantly altered, Akt phosphorylation is reduced by IFN-γ pretreatment and reraised by additional EGF, thus correlating Clt-dependent wound closure in scratch assays. G: Correlation of pAkt/Akt with mean wound closure in the second to fourth hour of the scratch assays in controls and Clt-involving constellations. Relative phosphorylation and wound closure rates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147736#pone.0147736.s005" target="_blank">S1 Table</a>) were plotted against each other and a regression line was computed. Pearson’s coefficient is 0.942 (p = 0.005).</p

Potassium channel-dependent wound healing of IEC-18 with and without 5 nm EGF under baseline or IFN-γ pretreated conditions.

<p>Wound healing of IEC-18 within six hours after mechanical injury. A: Influence of EGF on intestinal epithelial wound healing (n = 5). EGF increases wound healing both under baseline and inflammatory conditions (left panel). Right panel: Time course of restitution upon EGF treatment compared to control showing marked differences in the second, third and fourth hour after wounding. B: Impact of Clt with and without additional EGF after or without IFN-γ pretreatment (n = 5). EGF does not further increase Clt-dependent wound healing under baseline conditions, but fully reverts the Clt-dependent reduction in wound closure after proinflammatory treatment. C: Impact of 1-EBIO with and without additional EGF after or without IFN-γ pretreatment (n = 3–5). EGF promotes 1-EBIO-mediated wound healing both under baseline and inflammatory conditions.</p

Differential regulation of KCNN4-dependent intestinal epithelial wound healing in response to preincubation with IFN-γ.

<p>Wound healing of IEC-18 within six hours after mechanical injury. A: Comparison of wound healing of IEC-18 under control conditions without and with 24 h-pretreatment with IFN-γ (n = 5). Both the mean wound closure over six hours (left panel) and the time course profile of wound healing (right panel) are similar. B: Wound healing of IEC-18 after IFN-γ pretreatment and incubation with Clt and 1-EBIO (n = 5). KCNN4-inhibition by Clt results in significantly reduced rates of wound closure (left panel). Compared to Clt treatment without IFN-γ preincubation wound closure rates upon addition of Clt after 24h IFN-γ are significantly reduced in the second, third and fourth hour (right panel). C: Comparison of 1-EBIO-dependent wound closure to 1% DMSO (left panel, n = 5) and time course of 1-EBIO-dependent restitution with and without IFN-γ preincubation (right panel, n = 5). After proinflammatory treatment, 1-EBIO-dependent wound closure is higher than under baseline conditions.</p

Regulation of intestinal epithelial wound healing by potassium channel modulation.

<p>Wound healing of IEC-18 within six hours after mechanical injury. A: Representative images of wounds 0 and 6 hours after wounding in the presence of the indicated potassium channel modulators. Scale bar: 100μm. B: Quantitative analysis of wound healing of IEC-18 incubated with different potassium channel modulators (n = 5). While IbTx and Clt cause an increase in intestinal epithelial wound healing response, wound closure is significantly reduced after administration of 1-EBIO. C-E: Time course of wound healing after administration of IbTx, Clt and 1-EBIO (n = 5). F: Comparison of different solvent concentration applied (n = 5). While 0.25% (v/v) DMSO does not affect wound closure, 1% (v/v) DMSO significantly retards wound closure. G: Direct comparison of 1-EBIO with its solvent (n = 5). Reduction in wound healing by 1-EBIO is also significant vs. 1% DMSO.</p

KCNN4 mRNA expression in IBD.

<p>A: Relative levels of KCNN4 mRNA in IEC from controls (n = 10), UC (n = 8) and CD (n = 14). In both UC and CD the expression is increased vs. control. B: KCNQ1 mRNA levels are equal in UC (n = 5), CD (n = 11) and controls (n = 10) C: Comparison of KCNN4 expression in inflamed vs. uninflamed tissue of patients with CD. Left panel: Relative KCNN4 mRNA levels in all available samples (n = 14 inflamed, n = 7 not inflamed). Right panel: Comparison of KCNN4 levels in inflamed and uninflamed samples originating from the same patients (n = 5).</p

Image_3_Nr4a1-dependent non-classical monocytes are important for macrophage-mediated wound healing in the large intestine.tif

IntroductionMacrophages play an important role in intestinal wound healing. However, the trajectories from circulating monocytes to gut macrophages are incompletely understood.MethodsTaking advantage of mice depleted for non-classical monocytes due to deficiency for the transcription factor Nr4a1, we addressed the relevance of non-classical monocytes for large intestinal wound healing using flow cytometry, in vivo wound healing assays and immunofluorescence.ResultsWe show that wound healing in Nr4a1-deficient mice is substantially delayed and associated with reduced peri-lesional presence of macrophages with a wound healing phenotype.DiscussionOur data suggest that non-classical monocytes are biased towards wound healing macrophages. These insights might help to understand, how targeting monocyte recruitment to the intestine can be used to modulate intestinal macrophage functions.</p

Image_4_Healing of the epithelial barrier in the ileum is superior to endoscopic and histologic remission for predicting major adverse outcomes in ulcerative colitis.TIFF

BackgroundAchieving endoscopic remission is a key therapeutic goal in patients with ulcerative colitis (UC) that is associated with favorable long-term disease outcomes. Here, we prospectively compared the predictive value of endoscopic and/or histologic remission against ileal barrier healing for predicting long-term disease behavior in a large cohort of UC patients in clinical remission.MethodsAt baseline, UC patients in clinical remission underwent ileocolonoscopy with assessment of ileal barrier function by confocal endomicroscopy. Endoscopic and histologic disease activity and ileal barrier healing were scored using validated scores. During subsequent follow-up (FU), patients were closely monitored for clinical disease activity and occurrence of major adverse outcomes (MAO) defined as the following: disease relapse; UC-related hospitalization; UC-related surgery; necessity for initiation or dose escalation of systemic steroids, immunosuppressants, small molecules or biological therapy.ResultsOf the 73 UC patients included, 67% experienced MAO during a mean FU of 25 months. The probability of MAO-free survival was significantly higher in UC patients with endoscopic and/or histologic remission compared to patients with endoscopically and/or histologically active disease. Ileal barrier healing on endomicroscopy was highly accurate for predicting the further course of UC and outcompeted endoscopic and histologic remission for predicting MAO-free survival.ConclusionIleal barrier healing in clinically remittent UC patients can accurately predict future MAO development and is superior in its predictive capabilities than endoscopic and histologic remission. Ileal barrier healing therefore represents a novel and superior surrogate parameter for stratification of UC patients according to their risk for development of complicated disease behavior.Clinical trial registrationhttps://classic.clinicaltrials.gov/ct2/show/NCT05157750, identifier NCT05157750.</p