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. 2018 Feb 8;8(1):2619.
doi: 10.1038/s41598-018-20938-5.

Ileal pouch of ulcerative colitis and familial adenomatous polyposis patients exhibit modulation of autophagy markers

Nielce Maria Paiva  1 , Lívia Bitencourt Pascoal  1 , Leandro Minatel Vidal Negreiros  1 , Mariana Portovedo  2 , Andressa Coope  1 , Maria de Lourdes Setsuko Ayrizono  1 , Claudio Saddy Rodrigues Coy  1 , Marciane Milanski  2 , Raquel Franco Leal  3
Affiliations

Ileal pouch of ulcerative colitis and familial adenomatous polyposis patients exhibit modulation of autophagy markers

Nielce Maria Paiva et al. Sci Rep. .

Abstract

Total retocolectomy with ileal pouch-anal anastomosis (IPAA) is the surgery of choice for patients with ulcerative colitis (UC) that are refractory to clinical treatment. Pouchitis is one of the most common complications after this procedure. Defects in autophagy have been reported in inflammatory bowel diseases. However, there are no studies on the IP. Therefore, we studied markers for autophagy in the IP mucosa of UC and FAP patients comparing them to controls with a normal distal ileum. Sixteen patients with IP in "J" shape, asymptomatic and with endoscopically normal IP were evaluated. The control group consisted of eight patients with normal colonoscopy. There was a significant decrease in the transcriptional levels of ATG5, MAP1LC3A and BAX in the FAP group. There was also a decrease in the protein level of Beclin-1 in the UC and FAP compared to the control group. Although the LC3II levels by immunoblot were higher in the UC group, LC3/p62 co-localization were lower in the immunofluorescence analysis in the UC and FAP compared to the control group. Corroborating these results, there was an increase of p62 by immunoblot in the UC group. These findings indicated a modulation of macroautophagy markers in the IP, which may explain the mucosa inflammation predisposition.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Haematoxylin and Eosin (H&E) staining of ileal pouch mucosa biopsy of representatives Familial Adenomatous Polyposis (FAP) and Ulcerative Colitis (UC) patients. (A) Ileal pouch mucosa of a normal control (CTR Group). (B) Ileal pouch mucosa of FAP patient (FAP Group). (C) Ileal pouch mucosa of UC patient (UC Group). (D) Polymorphonuclear (PMN) leukocyte number of the lamina propria in the CTR, FAP and UC Groups. There is no statistical difference among the groups. Nuclear counterstaining: Mayer’s haematoxylin. Original magnification X20.
Figure 2
Figure 2
Evaluation of autophagy and apoptosis related gene expressions in the ileal pouch mucosa of Familial Adenomatous Polyposis (FAP) and Ulcerative Colitis (UC) patients. Transcriptional analysis reveals autophagy markers modulation in the ileal pouch mucosa of FAP patients. mRNA levels (qRT-PCR) of ULK1 (A), BECN1 (B), ATG16L1 (C), ATG5 (D), MAP1LC3A (E), BAX (F) and BCL2 (G) in ileal pouch mucosa of controls (CTR Group), FAP patients (FAP Group) and UC patients (UC Group). For FAP, n = 8; for UC, n = 8; for CTR, n = 8; *p < 0.05, **p < 0.01 and ***p < 0.001.
Figure 3
Figure 3
Evaluation of autophagy gene expressions in the ileal pouch afferent limb mucosa of Familial Adenomatous Polyposis (FAP) and Ulcerative Colitis (UC) patients. Transcriptional analysis reveals no differences compared to the controls. mRNA levels (qRT-PCR) of ULK1 (A), BECN1 (B), ATG16L1 (C), ATG5 (D), and MAP1LC3A (E) in ileal pouch afferent limb mucosa of controls (CTR Group), FAP patients (FAP-AF Group) and UC patients (UC-AF Group). For FAP-AF, n = 8; for UC-AF, n = 8; for CTR, n = 8; *p < 0.05, **p < 0.01 and ***p < 0.001.
Figure 4
Figure 4
Ileal pouch mucosa of Familial Adenomatous Polyposis (FAP) and Ulcerative Colitis (UC) patients shows autophagy protein markers modulation. Western blot analysis of Beclin-1 (A), LC3 (B), p62 (C) and HSC-70 (D) in ileal pouch (FAP and UC Groups) and in its afferent limb mucosa (FAP-AF and UC-AF Groups) of FAP and UC patients compared to controls (CTR Group). Each band represents one patient. For FAP, n = 8; for UC, n = 8; for CTR, n = 8; for FAP-AF, n = 8; for UC-AF, n = 8; *p < 0.05, **p < 0.01 and ***p < 0.001. ASU: arbitrary scanning unit.
Figure 5
Figure 5
Immunofluorescence staining of LC3 and p62 co-localization in the ileal pouch mucosa of Familial Adenomatous Polyposis (FAP) and Ulcerative Colitis (UC) patients. (A) Quantitative analysis of immunofluorescence staining for LC3 and p62 co-localized in FAP, UC and control (CTR) groups. (B) Representative staining of fixed paraffin-embedded ileal mucosa from the CTR, FAP and UC groups, showing low number of positive cells in FAP and UC groups compared to the CTR group. Positive cells are shown in orange (co-labeled by PI and FITC; overlay image) or red and green in the same cytosol (co-labeled by Alexa Fluor® 488 and Cy3®). Nucleus was stained with DAPI (blue-fluorescent). The arrows show the positive cells. Images were obtained using a 40X objective. For FAP, n = 8; for UC, n = 8; for CTR, n = 8; *p < 0.05, **p < 0.01 and ***p < 0.001.
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