1. Volume 140, Issue 2, Pages 529-539.e3 (February 2011)
Level of Activation of the Unfolded Protein Response Correlates With Paneth Cell Apoptosis in Human Small Intestine Exposed to Ischemia/Reperfusion 
Joep Grootjans, Caroline M. Hodin, Jacco–Juri de Haan, Joep P.M. Derikx, Kasper M.A. Rouschop, Fons K. Verheyen, Ronald M. van Dam, Cornelis H.C. Dejong, Wim A. Buurman, Kaatje Lenaerts 
Gastroenterology 
Volume 140, Issue 2, Pages e3 (February 2011)
DOI: /j.gastro
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2. Figure 1
Human small intestinal I/R results in activation of the unfolded protein response. (A, left panel) Splicing of XBP1 mRNA was observed upon reperfusion of ischemic jejunum. Band intensity of XBP1s was low in the 30I group, whereas significantly increased XBP1 splicing was observed in the 45I and 60I groups (representative XBP1 splicing assay of 3 patients per I/R group). (A, right panel) Quantification of the XBP1s/XBP1u ratio shows significant UPR activation after 30R in all I/R groups (*P < .001, compared with control). The XBP1s/XBP1u ratio was significantly higher after 45I 30R and 60I 30R as compared with 30I 30R (n = 10 per I/R group, P < .05 and P < .001, respectively). (B) Increased BiP staining (green) indicated UPR activation in crypt cells exposed to ischemia with 30R (arrows). Double staining for BiP (green) and lysozyme (red) identified these crypt cells as Paneth cells (right panel). (C) After 30R, up-regulation of potentially proapoptotic factor CHOP was observed in all I/R groups (*P < .001 compared with control, #P < .05 compared with control). Sustained CHOP expression was only observed at 45I 120R and 60I 120R (P < .05 compared to 30I 120R). (D) In line with CHOP expression, increased GADD34 expression was observed at 30R in all I/R groups (*P < .001). GADD34 expression was significantly higher after 60I 30R compared with 30I 30R (P < .05). In addition, GADD34 expression remained high at 120R in the 60I group, whereas it returned to baseline levels in the 30I group (P < .01). (E) Simultaneously with up-regulation of potentially proapoptotic UPR factors, double staining for lysozyme (blue, Paneth cell) and M30 (brown, apoptosis) as well as human defensin-5 (HD-5, blue, Paneth cell) and activated caspase-3 (Casp-3, brown, apoptosis) identified Paneth cell apoptosis (representative picture from tissue exposed to ischemia with 30R). (F) Double staining for BiP (green) and activated caspase-3 (red) showed that apoptotic Paneth cells exhibited signs of UPR activation.
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3. Figure 2
Prolonged human small intestinal I/R results in Paneth cell apoptosis. (A) Double staining for lysozyme (blue, Paneth cells) and M30 (brown, apoptosis) showed that Paneth cell apoptosis was absent in control jejunum not exposed to intestinal I/R. Paneth cell apoptosis was hardly observed after 30I with reperfusion (upper panel), whereas it became evident after longer ischemic periods (45I and 60I) with reperfusion (arrows, middle and lower panels). In addition, M30 staining was observed in enterocytes at the villus tips. (B) Quantification of the number of apoptotic Paneth cells showed significant Paneth cell apoptosis in jejunum exposed to ischemia with 30R compared with control (n = 10 per I/R group, *P < .01). Significantly more Paneth cell apoptosis occurred after 45I 30R and 60I 30R as compared with 30I 30R. (C) Apoptosis was rarely observed in other crypt cells (approximately 1% per crypt), indicating that Paneth cells were more susceptible to I/R-induced crypt cell apoptosis.
Gastroenterology  , e3DOI: ( /j.gastro )
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4. Figure 3
The number of apoptotic Paneth cells strongly correlates with the extent of UPR activation in the human small intestine. (A) XBP1s/XBP1u ratios, reflecting the extent of UPR activation, significantly correlated with the number of apoptotic Paneth cells at 30R (r = 0.85, P < .0001). (B) Double staining for apoptosis marker M30 and Paneth cell marker lysozyme showed more Paneth cell apoptosis in I/R-exposed intestine exhibiting high levels of spliced XBP1 mRNA.
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5. Figure 4
Shedding of apoptotic Paneth cells results in decreased Paneth cell numbers. (A) In longitudinal sections (left panel), detachment of apoptotic Paneth cells was observed at 30R (arrow, dashed line demarcates luminal side of crypt cells). Transverse sections (right panel) showed the presence of shed, apoptotic Paneth cells in the intestinal lumen. (B) Shedding of apoptotic Paneth cells resulted in excavation of small intestinal crypts at 120R (arrow). (C) Quantification of Paneth cells per crypt revealed a significant reduction of Paneth cell numbers after 60I 120R (n = 10 per I/R group, P < .001).
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6. Figure 5
Rat intestinal I/R induces UPR activation. (A) H&E staining showed villus sloughing at 60I and 60I 30R, whereas recovery was already observed at 120R. Debris of I/R-damaged cells was observed in the crypt lumen (white arrowheads). (B) XBP1 splicing assay revealed splice products during reperfusion of ischemically damaged jejunum (representative subset of 3 rats). (C) XBP1s/XBP1u ratio and (D) CHOP expression showed significant UPR activation at 60I 30R (P < .01 and P < .05, respectively) and 60I 120R (P < .05 and P < .05, respectively).
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7. Figure 6
Rat intestinal I/R induces ER stress and apoptosis of Paneth cells, resulting in lower numbers of Paneth cells. (A) Electron microscopy revealed normal appearance of the nucleus (N) and the rough ER (white arrowheads) in control jejunum, while most of the ER in shed Paneth cells at 60I 30R had disappeared and contracted. Heterochromatin condensation and nuclear fragmentation (blue arrowhead) showed Paneth cell apoptosis (blue dashed line demarcates crypts, red dashed line demarcates luminal side). G, granule; L, lumen. (B) TUNEL (green) and lysozyme (red) double staining confirmed apoptosis of Paneth cells (white arrowheads). (C) Lysozyme staining (red) showed a clear reduction of Paneth cells in I/R-exposed crypts. (D) Quantification of the number of lysozyme-positive cells revealed a 69.8% reduction of Paneth cells at 60I 120R (P < .01).
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8. Figure 7
Paneth cells play a crucial role in limiting bacterial translocation and inflammation during intestinal barrier integrity loss. (A) Hemorrhagic shock–induced physical barrier damage was observed in villus tips (*) of both rats with and without Paneth cell dysfunction. Immunohistochemistry for lysozyme shows Paneth cells (arrow) in non–Paneth cell dysfunction animals (upper panel) in both the healthy situation and following hemorrhagic shock–induced physical barrier damage. In animals with Paneth cell dysfunction, lysozyme staining was hardly observed (lower panel, arrowheads). (B) Paneth cell dysfunction simultaneous with physical intestinal damage resulted in increased bacterial translocation to MLN (P < .01), liver (P < .01), and spleen (P < .01) compared with non–Paneth cell dysfunction animals. (C) In addition, more vigorous inflammatory responses were observed in animals with Paneth cell dysfunction compared with controls, as reflected by both increased circulating tumor necrosis factor α levels (P < .01) and interleukin-6 levels (P < .01). CFU, colony forming units; PBD, physical barrier damage; PCD, Paneth cell dysfunction.
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9. Supplementary Figure 1
Hemorrhagic shock does not affect Paneth cells. Double staining for apoptosis (TUNEL, green) and Paneth cells (lysozyme, red) showed absence of apoptosis of Paneth cells in the rat small intestine exposed to 90 minutes of hemorrhagic shock (arrows) and a normal distribution of Paneth cells.
Gastroenterology  , e3DOI: ( /j.gastro )
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10. Supplementary Figure 2
Administration of dithizone results in selective Paneth cell loss. (A) Decreased lysozyme staining was observed in the small intestine of animals with Paneth cell dysfunction (arrows) compared with controls. (B) Lysozyme Western blot showed significantly decreased lysozyme protein expression in small intestinal tissue of animals with Paneth cell dysfunction compared with controls (P < .05). AU, arbitrary units. (C) H&E and zonula-occludens-1 staining showed similar intestinal morphology and tight junction status of animals with Paneth cell dysfunction and controls.
Gastroenterology  , e3DOI: ( /j.gastro )
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