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Autophagy Stimulation as a Potential Strategy Against Intestinal Fibrosis

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Abstract and Figures

We recently observed reduced autophagy in Crohn’s disease patients and an anti-inflammatory effect of autophagy stimulation in murine colitis, but both anti- and pro-fibrotic effects are associated with autophagy stimulation in different tissues, and fibrosis is a frequent complication of Crohn’s disease. Thus, we analyzed the effects of pharmacological modulation of autophagy in a murine model of intestinal fibrosis and detected that autophagy inhibition aggravates, while autophagy stimulation prevents, fibrosis. These effects are associated with changes in inflammation and in collagen degradation in primary fibroblasts. Thus, pharmacological stimulation of autophagy may be useful against intestinal fibrosis.
Correlation matrixes representing the Pearson's correlation coefficient between pairs of data on mRNA expression in intestinal explants. Murine intestinal fibrosis was induced by the heterotopic transplant of colonic tissue in mice receiving a daily intraperitoneal injection of the autophagy stimulator rapamycin (1.25 mg/kg mice), the autophagy inhibitor 3-methyladenine (3-MA, 10 mg/kg mice) or their vehicle (DMSO 1%, n = 5 each). The intestinal grafts resected seven days after transplantation were analyzed with regard to their mRNA expression of genes involved in i) fibrosis Figure 2. Correlation matrixes representing the Pearson's correlation coefficient between pairs of data on mRNA expression in intestinal explants. Murine intestinal fibrosis was induced by the heterotopic transplant of colonic tissue in mice receiving a daily intraperitoneal injection of the autophagy stimulator rapamycin (1.25 mg/kg mice), the autophagy inhibitor 3-methyladenine (3-MA, 10 mg/kg mice) or their vehicle (DMSO 1%, n = 5 each). The intestinal grafts resected seven days after transplantation were analyzed with regard to their mRNA expression of genes involved in (i) fibrosis and epithelial to mesenchymal transition; and (ii) inflammation: the general macrophage marker (F4/80), pro-inflammatory (red), anti-inflammatory (blue) and pro-fibrotic (purple) molecules. The data were organized taking as references: (i) the fibroblast marker vimentin (in all groups); and (ii) the macrophage marker which correlates with a higher number of markers of inflammation in each experimental group (vehicle: CD163; rapamycin: F4/80, 3-MA: CD16). The pairwise comparison heatmaps were performed using the online resource available at http://www.heatmapper.ca, and interpreted according to its authors' instructions [7].
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cells
Brief Report
Autophagy Stimulation as a Potential Strategy
Against Intestinal Fibrosis
Jesus Cosin-Roger 1,2, Francisco Canet 3, Dulce C. Macias-Ceja 3, Laura Gisbert-Ferrándiz 3,
Dolores Ortiz-Masiá2,4, Juan V. Esplugues 1,2,3, Rafael Alós5, Francisco Navarro 6,
María D. Barrachina 2, 3, * and Sara Calatayud 2,3
1Hospital Dr Peset, FISABIO, 46017 Valencia, Spain
2
Centro de Investigaci
ó
n Biom
é
dica en Red de Enfermedades Hep
á
ticas y Digestivas, 46010 Valencia, Spain
3Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, 46010 Valencia, Spain
4Departamento de Medicina, Facultad de Medicina, Universidad de Valencia, 46010 Valencia, Spain
5Departamento de Cirugía del Aparato Digestivo, Hospital La Fe, 46526 Valencia, Spain
6Departamento de Cirugía y Coloproctología, Hospital de Manises, 46940 Valencia, Spain
*Correspondence: dolores.barrachina@uv.es
Received: 13 August 2019; Accepted: 11 September 2019; Published: 13 September 2019


Abstract:
We recently observed reduced autophagy in Crohn’s disease patients and an
anti-inflammatory eect of autophagy stimulation in murine colitis, but both anti- and pro-fibrotic
eects are associated with autophagy stimulation in dierent tissues, and fibrosis is a frequent
complication of Crohn’s disease. Thus, we analyzed the eects of pharmacological modulation
of autophagy in a murine model of intestinal fibrosis and detected that autophagy inhibition
aggravates, while autophagy stimulation prevents, fibrosis. These eects are associated with
changes in inflammation and in collagen degradation in primary fibroblasts. Thus, pharmacological
stimulation of autophagy may be useful against intestinal fibrosis.
Keywords: intestinal fibrosis; autophagy; inflammation
1. Introduction
Crohn’s Disease (CD) is a chronic inflammatory pathology of the gut that in a significant proportion
of patients, leads to complications related to the development of intestinal fibrosis and strictures that
often need a surgical intervention.
Genetic studies identified several single nucleotide polymorphisms (SNPs) associated with CD
in genes related to autophagy, a cellular process essential in intestinal homeostasis [
1
]. We reported
that autophagy is reduced in the damaged mucosa of CD patients [
2
] and that autophagy stimulation
prevents intestinal inflammation [
3
]. However, control of inflammation by the current therapies does
not seem enough to prevent fibrosis and both anti- and pro-fibrotic eects have been attributed to
autophagy stimulation in dierent organic systems [
4
,
5
]. Hence, we aim to analyze the eects of
pharmacological modulation of autophagy in the development of intestinal fibrosis.
2. Methods
2.1. Heterotopic Transplant Model of Intestinal Fibrosis
The murine intestinal fibrosis was induced
in vivo
by the heterotopic transplant of colonic tissue [
6
].
Briefly, colon resections of 1cm from C57BL/6 mice were washed with 0.9% NaCl and transplanted
subcutaneously into the neck of recipient mice. After anesthetizing the mice with isoflurane, two
perpendicular incisions to the body axis were made, the intestinal grafts were implanted and the
Cells 2019,8, 1078; doi:10.3390/cells8091078 www.mdpi.com/journal/cells
Cells 2019,8, 1078 2 of 8
incisions were closed with vicryl 5-stiches. We kept an adjacent segment of the colon from each
donor mice as autologous control tissue. Recipient mice were treated with a daily intraperitoneal
injection of Rapamycin (1.25 mg/kg), 3-methyladenine (3MA) (10 mg/kg) or its vehicle (DMSO
1%). Seven days after surgery, recipient mice were sacrificed by neck dislocation and intestinal
grafts were collected. All tissues were subdivided for RNA and protein isolation and histological
analysis. All protocols were approved by the institutional animal care and use committe of the
University of Valencia, and all experiments were performed in compliance with the European Animal
Research Laws (European Communities Council Directives 2010/63/EU, 90/219/EEC, Regulation (EC)
No. 1946/2003) and Generalitat Valenciana (Art
í
culo 31, Real Decreto 53/2013) (Ethical approval number
2018/VSC/PEA/0179, 28 August 2018).
2.2. Primary Intestinal Fibroblasts Isolation and Culture
Primary intestinal fibroblasts were obtained from the healthy tissue of intestinal resections from
colon carcinoma patients [
6
]. Briefly, intestinal tissue was cut into 3-5mm pieces and epithelial
cells were removed with an incubation with HBSS-EDTA of 30 min at 37
º
C. Subsequently, small
intestinal pieces were digested with collagenase I (1 mg/ml), DNAse (1
µ
l/ml) and hyaluronidase
(2 mg/ml) during 30 min at 37
º
C. Finally, explants were maintained under culture with DMEM high
glucose (Sigma-Aldrich) supplemented with FCS 20%, penicilin/streptomicin (100
µ
g/ml), gentamycin
(100
µ
g/ml), amphotericin B (2
µ
g/ml), and ciprofloxacin (16
µ
g/ml) in a Petri dish. Primary intestinal
fibroblasts were treated with TGF-
β
(5 ng/ml) and Rapamycin (50 nM) or Bafilomycin B1 (10 nM)
during 24 h. The study was approved by the Institutional Review Board of both Hospital of Manises
(Valencia, Spain) and Hospital La Fe (Valencia, Spain). Written informed consent was obtained from all
participating patients.
2.3. RNA Extraction and Quantitative PCR
RNA from intestinal grafts/tissues and primary fibroblasts was isolated using the Illustra RNA Spin
Mini (GE Healthcare). The tissue homogenization was performed with the gentleMACS
Dissociator
(Miltenyi Biotec), while primary fibroblasts were lysed with a 19G needle. cDNA was obtained after
RT-PCR performed with the Prime Script RT reagent Kit (Takara Biotechnology). The expression of
several genes (Table S1) was analyzed by quantitative PCR performed with the Prime Script Reagent
Kit Perfect Real Time (Takara Biotechnology). Relative gene expression of each gene was expressed as
follows: fold induction =2
(
CT), where
CT =CT (target)
CT (housekeeping), and
(
CT) =
CT (treated) CT (control). In all cases β-actin was used as the housekeeping gene.
2.4. Western Blot
Equal amounts of protein from intestinal grafts or primary intestinal fibroblasts were loaded onto
SDS-PAGE gels. After electrophoresis and transference, membranes were blocked with 5% non-fat
dry milk in TBS-T during 1 h at room temperature and incubated overnight at 4
C with the primary
antibody (Table S2). Afterwards, membranes were washed with TBS-T and incubated with a secondary
antibody anti-mouse IgG (Thermo Scientific, 1:2500) or anti-rabbit IgG (Thermo Scientific, 1:5000)
during 1 h at room temperature. Protein bands were detected by LAS-300 (Fujifilm) after treatment
with SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific). The densitometry of the
bands was quantified with the software Image Gauge Version 4.0 (Fujifilm).
2.5. Sirius Red Staining
Sirius Red Staining was performed in 5
µ
m sections of paran-embedded colonic tissues in order
to analyze the collagen layer in intestinal grafts obtained after the heterotopic transplant model. After
deparanization and rehydration, slides were incubated with Fast green (Sigma-Aldrich) during
15 min at room temperature and with Sirius red 0.1% (Sigma-Aldrich) /Fast green 0.04% during 30 min
Cells 2019,8, 1078 3 of 8
at room temperature. Finally, tissues were dehydrated and visualized with a light microscope (1X81
Olympus).
2.6. Statistical Analysis
All data were expressed as mean
±
S.E.M. and were compared by analysis of variance (one-way
ANOVA) with a Newman–Keuls post hoc correction for multiple comparisons or a t-test when
appropriate (Graph-Pad Software 6.0). A p-value <0.05 was considered to be statistically significant.
3. Results
Seven days after implantation, grafts exhibited a significant inhibition of autophagy as shown by
the accumulation of p62 and the reduction of LC3II and Beclin-1 (Figure 1A). As expected, rapamycin
stimulated and 3-MA further inhibited autophagy in the grafted tissue (Figure 1B).
Explanted colon tissues presented a significant deposition of collagen in the mucosa, submucosa
and subserosa. Of interest, treatment of mice with rapamycin significantly reduced the collagen
layer thickness and the contrary occurred in tissues resected from 3-MA-treated mice (Figure 1C).
These results were strongly reinforced by the gene expression of pro-fibrotic markers. The fibrotic
grafts from vehicle-treated mice showed a significantly increased expression of the two fibrous
collagens (Col1a1, Col3a1) (Figure 1D), and of other molecules associated with fibrosis and epithelial
to mesenchymal transition (Vimentin, TGFß, Timp1, Mmp2, Snail1, Snail2, Itgb6) with a parallel
reduction in the epithelial marker E-cadherin. Grafts from mice receiving rapamycin showed reduced
mRNA expression of these collagens and a partial reversion on the E-cadherin reduction, while
the other parameters were not significantly modified. Finally, tissues from 3-MA-treated animals
showed a significant up-regulation of most pro-fibrotic genes with regard to the expression observed
in vehicle-treated mice (Figure 1D,E).
Cells 2019, 8, x 4 of 9
Figure 1. Murine fibrosis is reduced by autophagy stimulation and increased by autophagy inhibition.
Murine intestinal fibrosis was induced by the heterotopic transplant of colonic tissue. Autophagy and
fibrotic markers were analyzed in intestinal control tissues and the intestinal grafts resected seven
days after transplantation from mice receiving a daily intraperitoneal injection of the autophagy
stimulator rapamycin (1.25 mg/kg mice), the autophagy inhibitor 3-Methyladenine (3-MA, 10 mg/kg
mice) or their vehicle (DMSO 1%, n = 5 each). (A) Representative Western blots showing the protein
levels of the autophagy substrate P62, the autophagy protein Beclin-1, the two forms of LC3 (LC3-I,
cytoplasmic; LC3-II, autophagosome-associated) and of Glyceraldehyde-3-Phosphate
Dehydrogenase (GAPDH). The accumulation of P62 and the reduction in Beclin-1 and LC3-II in the
explants from vehicle-treated mice indicate an inhibited autophagy that was partially prevented by
rapamycin- and promoted by 3-MA- treatments (B). (C) Representative pictures of Sirius Red staining
in paraffin-embedded tissues where the red color indicates collagen deposition (20× and 40×
magnification in upper and lower panels respectively), and (D) mRNA expression of fibrous collagens
(Col1a1, Col3a1) analyzed by qPCR (results normalized with β-actin and represented as fold induction
vs. intestinal control tissues). (E) Heatmap showing the relative mRNA expression of genes involved
Figure 1. Cont.
Cells 2019,8, 1078 4 of 8
Cells 2019, 8, x 4 of 9
Figure 1. Murine fibrosis is reduced by autophagy stimulation and increased by autophagy inhibition.
Murine intestinal fibrosis was induced by the heterotopic transplant of colonic tissue. Autophagy and
fibrotic markers were analyzed in intestinal control tissues and the intestinal grafts resected seven
days after transplantation from mice receiving a daily intraperitoneal injection of the autophagy
stimulator rapamycin (1.25 mg/kg mice), the autophagy inhibitor 3-Methyladenine (3-MA, 10 mg/kg
mice) or their vehicle (DMSO 1%, n = 5 each). (A) Representative Western blots showing the protein
levels of the autophagy substrate P62, the autophagy protein Beclin-1, the two forms of LC3 (LC3-I,
cytoplasmic; LC3-II, autophagosome-associated) and of Glyceraldehyde-3-Phosphate
Dehydrogenase (GAPDH). The accumulation of P62 and the reduction in Beclin-1 and LC3-II in the
explants from vehicle-treated mice indicate an inhibited autophagy that was partially prevented by
rapamycin- and promoted by 3-MA- treatments (B). (C) Representative pictures of Sirius Red staining
in paraffin-embedded tissues where the red color indicates collagen deposition (20× and 40×
magnification in upper and lower panels respectively), and (D) mRNA expression of fibrous collagens
(Col1a1, Col3a1) analyzed by qPCR (results normalized with β-actin and represented as fold induction
vs. intestinal control tissues). (E) Heatmap showing the relative mRNA expression of genes involved
Figure 1.
Murine fibrosis is reduced by autophagy stimulation and increased by autophagy inhibition.
Murine intestinal fibrosis was induced by the heterotopic transplant of colonic tissue. Autophagy
and fibrotic markers were analyzed in intestinal control tissues and the intestinal grafts resected
seven days after transplantation from mice receiving a daily intraperitoneal injection of the autophagy
stimulator rapamycin (1.25 mg/kg mice), the autophagy inhibitor 3-Methyladenine (3-MA, 10 mg/kg
mice) or their vehicle (DMSO 1%, n=5 each). (
A
) Representative Western blots showing the
protein levels of the autophagy substrate P62, the autophagy protein Beclin-1, the two forms of
LC3 (LC3-I, cytoplasmic; LC3-II, autophagosome-associated) and of Glyceraldehyde-3-Phosphate
Dehydrogenase (GAPDH). The accumulation of P62 and the reduction in Beclin-1 and LC3-II in
the explants from vehicle-treated mice indicate an inhibited autophagy that was partially prevented
by rapamycin- and promoted by 3-MA- treatments (
B
). (
C
) Representative pictures of Sirius Red
staining in paran-embedded tissues where the red color indicates collagen deposition (20
×
and
40
×
magnification in upper and lower panels respectively), and (
D
) mRNA expression of fibrous
collagens (Col1a1, Col3a1) analyzed by qPCR (results normalized with
β
-actin and represented as
fold induction vs. intestinal control tissues). (
E
) Heatmap showing the relative mRNA expression of
genes involved in (i) fibrosis and epithelial to mesenchymal transition; (ii) inflammation: the general
macrophage marker (F4/80), pro-inflammatory (red), anti-inflammatory (blue) and pro-fibrotic (purple)
molecules; and (iii) T lymphocyte markers (regulatory T cells—green, Th17—orange). Bars in graphs
represent
mean ±S.E.M.
Significant dierences vs. control intestinal tissues are shown by * p<0.05,
** p<0.01 and
*** p<0.001
; significant dierences vs. grafts obtained from vehicle-treated mice are
shown by
++ p<0.01
; as analyzed by ANOVA with a Newman–Keuls post hoc correction for multiple
comparisons (Graph-Pad Software v6.0).
In line with our previous study [
6
], grafts obtained from vehicle-treated mice showed an increased
expression of macrophage markers, cytokines and other modulators of inflammation. We observed an
up-regulation of pro-inflammatory (red), anti-inflammatory (blue) and pro-fibrotic (purple) molecules.
These tissues also presented an increased expression of T lymphocyte markers (regulatory T cells—green,
Th17—orange). Rapamycin treatment increased macrophage infiltration (F4/80 up-regulation) and
the expression of anti-inflammatory agents. On the contrary, 3-MA promoted the expression of
pro-inflammatory and pro-fibrotic mediators as well as that of T cell markers (Figure 1E). From the
analysis of these results in a correlation matrix we deduce that in 3-MA treated mice, CD16-expression
is associated with that of several markers of regulatory/pro-fibrotic macrophages that in turn, correlate
with the expression of most fibrotic indicators. The definition of a predominant macrophage phenotype
associated with fibrosis is less clear in animals treated with vehicle, and completely absent in
rapamycin-treated animals (Figure 2).
Cells 2019,8, 1078 5 of 8
Cells 2019, 8, x 6 of 9
Figure 2. Correlation matrixes representing the Pearson’s correlation coefficient between pairs of data
on mRNA expression in intestinal explants. Murine intestinal fibrosis was induced by the heterotopic
transplant of colonic tissue in mice receiving a daily intraperitoneal injection of the autophagy
stimulator rapamycin (1.25 mg/kg mice), the autophagy inhibitor 3-methyladenine (3-MA, 10 mg/kg
mice) or their vehicle (DMSO 1%, n = 5 each). The intestinal grafts resected seven days after
transplantation were analyzed with regard to their mRNA expression of genes involved in i) fibrosis
Figure 2.
Correlation matrixes representing the Pearson’s correlation coecient between pairs of
data on mRNA expression in intestinal explants. Murine intestinal fibrosis was induced by the
heterotopic transplant of colonic tissue in mice receiving a daily intraperitoneal injection of the
autophagy stimulator rapamycin (1.25 mg/kg mice), the autophagy inhibitor 3-methyladenine (3-MA,
10 mg/kg mice) or their vehicle (DMSO 1%, n=5 each). The intestinal grafts resected seven days
after transplantation were analyzed with regard to their mRNA expression of genes involved in (i)
fibrosis and epithelial to mesenchymal transition; and (ii) inflammation: the general macrophage
marker (F4/80), pro-inflammatory (red), anti-inflammatory (blue) and pro-fibrotic (purple) molecules.
The data were organized taking as references: (i) the fibroblast marker vimentin (in all groups); and
(ii) the macrophage marker which correlates with a higher number of markers of inflammation in
each experimental group (vehicle: CD163; rapamycin: F4/80, 3-MA: CD16). The pairwise comparison
heatmaps were performed using the online resource available at http://www.heatmapper.ca, and
interpreted according to its authors’ instructions [7].
Cells 2019,8, 1078 6 of 8
Of interest, in human primary fibroblasts treated with TGF-
β
, the increase in autophagy induced
by rapamycin was associated with a reduction in the Col1a1 protein while autophagy blockade with
bafilomycin-B1 provoked its accumulation (Figure 3A). None of these treatments modified Col1a1
mRNA expression (Figure 3B).
Cells 2019, 8, x 7 of 9
data were organized taking as references: i) the fibroblast marker vimentin (in all groups); and, ii) the
macrophage marker which correlates with a higher number of markers of inflammation in each
experimental group (vehicle: CD163; rapamycin: F4/80, 3-MA: CD16). The pairwise comparison
heatmaps were performed using the online resource available at http://www.heatmapper.ca, and
interpreted according to its authors’ instructions [7].
Of interest, in human primary fibroblasts treated with TGF-β, the increase in autophagy induced
by rapamycin was associated with a reduction in the Col1a1 protein while autophagy blockade with
bafilomycin-B1 provoked its accumulation (Figure 3A). None of these treatments modified Col1a1
mRNA expression (Figure 3B).
Figure 3. Human primary fibroblasts isolated from the healthy margin of intestinal carcinoma
resections (n = 5) were treated with TGF-β (5ng/ml), in the presence of Rapamycin (50 nM),
Bafilomycin B1 (10 nM) or their vehicles, for 24 hours. (A) Representative Western blots and protein
levels of Col1a1, P62, LC3-I/II and GAPDH; and (B) graphs showing Col1a1 and P62 mRNA
expression (results normalized with β-actin and represented as fold induction vs. vehicle-treated
cells). Bars in graphs represent mean±S.E.M. Significant differences vs. vehicle-treated fibroblasts are
shown by *p < 0.05, and ***p < 0.001; as analyzed by ANOVA with a Newman–Keuls post hoc
correction for multiple comparisons or a t-test when appropriate (Graph-Pad Software v6.0).
Figure 3.
Human primary fibroblasts isolated from the healthy margin of intestinal carcinoma resections
(n=5) were treated with TGF-
β
(5 ng/ml), in the presence of Rapamycin (50 nM), Bafilomycin B1 (10 nM)
or their vehicles, for 24 h. (
A
) Representative Western blots and protein levels of Col1a1, P62, LC3-I/II and
GAPDH; and (
B
) graphs showing Col1a1 and P62 mRNA expression (results normalized with
β
-actin
and represented as fold induction vs. vehicle-treated cells). Bars in graphs represent
mean ±S.E.M.
Significant dierences vs. vehicle-treated fibroblasts are shown by * p<0.05, and
*** p<0.001
; as
analyzed by ANOVA with a Newman–Keuls post hoc correction for multiple comparisons or a t-test
when appropriate (Graph-Pad Software v6.0).
4. Discussion
This study demonstrates a reduced autophagy in murine intestinal fibrosis. Autophagy stimulation
exerts an anti-fibrotic eect and enhances collagen degradation in fibroblasts, while autophagy inhibition
aggravates fibrosis in association with significant changes in the inflammatory response and a reduction
in collagen digestion.
Fibrosis has been linked with both increased and defective autophagy in dierent organic
contexts [
4
,
5
]. In the intestine, SNPs in genes of autophagy predispose to inflammatory bowel
disease (IBD) and their presence is associated with the development of stricturing and penetrating
complications [
8
,
9
]. In coherence with this clinical data, the development of fibrosis in our murine
model is associated with an inhibition of autophagy and the contribution of a defective autophagy to
fibrosis is further substantiated by the worsening eect induced by the autophagy inhibitor 3-MA. Of
significance, the stimulation of autophagy with rapamycin prevents this pathological process.
Cells 2019,8, 1078 7 of 8
We observed that the inflammatory response that accompanied fibrosis was significantly aected
by autophagy modulation. In vehicle-treated animals, the increased macrophage infiltrate seems
to include both pro-inflammatory and anti-inflammatory/regulatory phenotypes. In contrast, in
3-MA-treated mice, we encountered an increased expression of pro-inflammatory mediators and a
well-defined macrophage phenotype characterized by CD16-expression that, as seen before [
10
], seems
relevant for the fibrotic process. These macrophages, together with CD16, express other classic M2
markers and are probably the source of the pro-fibrotic cytokines IL6, IL13, and IL8 [
11
,
12
]. The increase
in fibrosis may also be related to the higher infiltration of Th17 and regulatory T cells observed in
these tissues [
13
,
14
]. Finally, stimulation of autophagy with rapamycin increased the expression of
anti-inflammatory mediators, as occurred in colitis [
3
], and augmented the influx of macrophages that
seem of a regulatory/anti-inflammatory profile. In line with this, an anti-fibrotic eect of autophagy
stimulation and the consequent inhibition of the IL-22/IL-23 axis in macrophages has been recently
demonstrated in a dierent model of intestinal fibrosis [
15
]. Finally, we observed that treatment
of isolated primary fibroblasts with rapamycin or the autophagy blocker bafilomycin significantly
decreased or increased, respectively, collagen protein levels without aecting its gene expression. This
implies that fibroblast’s autophagy, and its pharmacological regulation, aects collagen degradation.
This mechanism most probably contributes to the eects of rapamycin and 3-MA on fibrosis
in vivo
and would add to the regulation of collagen gene expression observed in the colonic grafts. Thus,
autophagy stimulation seems to inhibit intestinal fibrosis by modulating the function of the innate
immune system and the mesenchymal activity.
In summary, autophagy inhibition seems relevant in the development of intestinal fibrosis and the
pharmacological activation of autophagy constitutes a promising strategy against this CD complication.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2073-4409/8/9/1078/s1,
Table S1. Primer sequences of specific PCR products for each gene analysed. Table S2. Primary antibodies used in
Western Blot analysis.
Author Contributions:
Conceptualization, S.C., M.D.B., J.C.R., J.V.E.; Methodology, F.C., D.C.M., L.G.F., D.O.M.;
Formal Analysis, S.C., M.D.B., J.C.R.; Investigation, F.C., D.C.M., L.G.F., D.O.M., R.A., F.N.; Resources, J.V.E.,
R.A., F.N.; Writing—Original Draft Preparation, S.C., J.C.R.; Writing—Review & Editing, S.C., J.C.R., M.D.B.;
Supervision, S.C., M.D.B.
Funding:
This work was supported by Ministerio de Econom
í
a y Competitividad and the European Regional
Development Fund of the European Union [ERDF] [SAF2013-43441P], Ministerio de Economia, Industria y
Competitividad and ERDF [SAF2016-80072P], CIBERehd [CB06/04/0071, CIBER-EHD 2016/ACCESS EHD16PI02,
CIBEREHD Investigadores Noveles EHD19PI05], Generalitat Valenciana [PROMETEOII/2014/035], [GV/2018/041]
and [PROMETEO/2018/141], the ECCO Pioneer Award and a research grant from the IOIBD.
Conflicts of Interest: The authors declare no conflict of interest.
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
... In a murine model of intestinal fibrosis, it has been demonstrated that autophagy inhibition aggravates, while autophagy stimulation prevents, fibrosis. 39 In addition to the 'in vitro' observation that fibroblast autophagy, and its pharmacological regulation, affects collagen degradation, the tissue inflammatory response that accompanied fibrosis was significantly affected by autophagy modulation. In particular, in mice treated with the autophagy inhibitor, an increased expression of pro-inflammatory mediators and pro-fibrogenic 40 CD16 + -M2 macrophage infiltration was observed, while stimulation of autophagy with rapamycin increased the expression in mice of anti-inflammatory mediators and infiltration of macrophages with a regulatory/anti-inflammatory profile. ...
... In particular, in mice treated with the autophagy inhibitor, an increased expression of pro-inflammatory mediators and pro-fibrogenic 40 CD16 + -M2 macrophage infiltration was observed, while stimulation of autophagy with rapamycin increased the expression in mice of anti-inflammatory mediators and infiltration of macrophages with a regulatory/anti-inflammatory profile. 39 Thus, autophagy, macrophages and related cytokine modulation seem to play a relevant role in the fibrogenic process. This view is reinforced by the observations made in the murine model of TNBS chronic colitis. ...
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Background and Aims Intestinal fibrosis is a common complication of inflammatory bowel diseases. Medical treatment of intestinal fibrosis is an unmet therapeutic need. CD147 overexpression can induce myofibroblast differentiation associated with extracellular matrix deposition, favouring the development of fibrosis. To understand whether CD147 may promote intestinal fibrosis, we analysed its expression and blocked its function by using its specific inhibitor AC-73 [3-{2-[([1,1’-biphenyl]-4-ylmethyl) amino]-1-hydroxyethyl} phenol] in the murine TNBS [trinitrobenzenesulfonic acid]-chronic colitis model associated with intestinal fibrosis. Methods TNBS chronic colitis was induced by weekly intrarectal administration of escalating doses of TNBS. Ethanol-treated and untreated mice were used as controls. Separated groups of TNBS, ethanol-treated or untreated mice received AC-73 or vehicle administered intraperitoneally from day 21 to day 49. At day 49, mice were killed, and colons collected for histological analysis, protein and RNA extraction. CD147, α-SMA and activated TGF-β1 protein levels, CD147/ERK/STAT3 signalling pathway and autophagy were assessed by Western blot, collagen and inflammatory/fibrogenic cytokines mRNA tissue content by quantitative PCR. Results In mice with chronic TNBS colitis, CD147 protein level increased during fibrosis development in colonic tissue, as compared to control mice. CD147 inhibition by AC-73 treatment reduced intestinal fibrosis, collagen and cytokine mRNA tissue content, without significant modulation of activated TGF-β1 protein tissue content. AC-73 inhibited CD147/ERK1/2 and STAT3 signalling pathway activation and induced autophagy. Conclusions CD147 is a potential new target for controlling intestinal fibrosis and its inhibitor, AC-73, might represent a potential new anti-fibrotic therapeutic option in IBD.
... Total RNA from cells and murine and human tissues was isolated using direct-zol RNA MiniPrep Plus R2072 from ZymoResearch according to the manufacturer's instructions. Mice tissue and intestinal resections were homogenated with TRI Reagent ® (ZymoResearch, Irvine, CA, USA), using the GentleMACS Dissociator (Milteny Biotech, Gladbach, Germany) as previously described [36]. cDNA was obtained from previously isolated RNA by reverse transcription PCRusing the the PrimeScript RT reagent Kit (Takara Bitechnology, Dalian, China). ...
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Intestinal epithelial cells (IECs) constitute a defensive physical barrier in mucosal tissues and their disruption is involved in the etiopathogenesis of several inflammatory pathologies, such as Ulcerative Colitis (UC). Recently, the succinate receptor SUCNR1 was associated with the activation of inflammatory pathways in several cell types, but little is known about its role in IECs. We aimed to analyze the role of SUCNR1 in the inflammasome priming and its relevance in UC. Inflammatory and inflammasome markers and SUCNR1 were analyzed in HT29 cells treated with succinate and/or an inflammatory cocktail and transfected with SUCNR1 siRNA in a murine DSS model, and in intestinal resections from 15 UC and non-IBD patients. Results showed that this receptor mediated the inflammasome, priming both in vitro in HT29 cells and in vivo in a murine chronic DSS-colitis model. Moreover, SUNCR1 was also found to be involved in the activation of the inflammatory pathways NFкB and ERK pathways, even in basal conditions, since the transient knock-down of this receptor significantly reduced the constitutive levels of pERK-1/2 and pNFкB and impaired LPS-induced inflammation. Finally, UC patients showed a significant increase in the expression of SUCNR1 and several inflammasome components which correlated positively and significantly. Therefore, our results demonstrated a role for SUCNR1 in basal and stimulated inflammatory pathways in intestinal epithelial cells and suggested a pivotal role for this receptor in inflammasome activation in UC.
... Our study also confirmed that proinflammatory markers were not correlated with fibrosis-associated markers, and preventive intervention did not achieve better anti-fibrotic efficacy. Defects in autophagy have been linked with fibrosis in different organs, especially in the intestine, and several mutated autophagy genes are associated with the development of structuring and penetrating complications in IBD [33,34]. Of note, the interaction between autophagy and EMT has been reported in other studies, with a recent study showing that autophagy inhibition induces EMT via the p62/SQSMT1-NFκB-Snail2 pathway in alveolar epithelial cells and promotes local myofibroblast differentiation through paracrine signals [23]. ...
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Background Intestinal fibrosis is the most common complication of inflammatory bowel disease; nevertheless, specific therapies are still unavailable. Resolvin D1 (RvD1), a typical endogenous ω-3 fatty acid-derived lipid mediator, has attracted wide attention due to its remarkable anti-fibrosis effects. However, the efficacy and mechanisms of RvD1 in intestinal fibrosis remain unclear.AimTo investigate the protective effect of RvD1 in a dextran sulfate sodium (DSS)-induced intestinal fibrosis model and explore the molecular mechanisms underlying its anti-fibrotic effect.MethodsA DSS-induced intestinal fibrosis model and intestinal epithelial-to-mesenchymal transition (EMT) model were used to observe the efficacy of RvD1, and fibroblasts were stimulated with conditioned medium with or without TGF-β1 to investigate the probable mechanisms of RvD1 in intestinal fibrosis disease.ResultsIntestinal fibrosis was effectively alleviated by RvD1 in a DSS-induced model, both preventively and therapeutically, and autophagy inhibition-induced EMT in intestinal epithelial cells was significantly suppressed in vivo and in vitro. Furthermore, RvD1 reduced epithelial cell EMT paracrine signaling, which promoted the differentiation of local fibroblasts into myofibroblasts.Conclusions Our results suggested that RvD1 reduces autophagy-induced EMT in intestinal epithelial cells and ameliorates intestinal fibrosis by disrupting epithelial–fibroblast crosstalk.
... Other studies observed that autophagy seems to inhibit intestinal fibrosis by modulating the function of the innate immune system and the mesenchymal activity. [3,12] (7) Balance intestinal epithelial cells (ISCs) regeneration. The critical role of autophagy in maintaining ISC functions under different physiological conditions has been discovered only in recent years. ...
... Autophagy, a 'self-digestion' process that aims to reuse cellular components to adapt to starvation and stress, participates in many aspects of biology [29]. Indeed, studies have demonstrated that autophagy is a potential target in preventing fibrosis [28,30]. Fibroblast proliferation and migration play a crucial role in arthrofibrosis. ...
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Chapter
The major distinguishing feature of fibrosis is significant deposition of collagen and other extracellular matrix (ECM) proteins, which can result in scarring if sufficiently excessive. Fibrosis affects many tissue types, and thus contributes to a broad group of diseases which, with few exceptions, continues to lack specific therapy. It has been estimated that nearly 45% of deaths in the developed world are caused by fibroproliferative diseases, which contribute to cardiovascular disease, pulmonary, renal, gut and liver fibrosis, and scleroderma (Bitterman and Henke in Chest 99:81S–84S, [1]). Fibroblasts are the most common stromal cell type of the connective tissues found in the body, and are the primary source of ECM in physiological conditions, i.e. in the absence of disease. The conversion of fibroblasts or similar stromal cells to myofibroblasts is a principal mediator of pathological fibrosis in many tissue types, and frequently occurs in response to ongoing tissue injury and chronic inflammation. While the fibrotic response can occur in response to existing disease, the phenotype conversion of fibroblasts to myofibroblasts due to transient stress or damage may lead to the initiation of long-term fibrotic disease (Bagchi et al. in BMC Biol 14:21, [2]). Inflammation has been found to be a critical inducer of fibrosis, with immune cells generating a variety of growth factors and cytokines that play critical roles in fibroblast activation and subsequent tissue remodelling and fibrosis. A common cellular response to stress stimuli such as inflammation is autophagy, and recent studies have tightly linked the activation or inhibition of autophagy with fibrotic diseases in myriad tissues. Here, we discuss the inter-relationship of these pathways to provide insight into their potential as therapeutic targets in fibrotic disease.
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Intestinal fibrosis is an excessive proliferation of myofibroblasts and deposition of collagen, a condition frequently seen in Crohn’s disease (CD). The mechanism underlying myofibroblast hyper-proliferation in CD needs to be better understood. In this report, we found that mTOR inhibitor rapamycin or mTOR deletion in CX3Cr1+ mononuclear phagocytes inhibits expression of interleukin (IL)−23, accompanied by reduced intestinal production of IL-22 and ameliorated fibrosis in the TNBS-induced fibrosis mouse model. This inhibition of IL-23 expression is associated with elevated autophagy activity. Ablating the autophagy gene Atg7 increases the expression of IL-23, leading to increased expression of IL-22 and increased fibrosis. Both induction of IL-22 and intestinal fibrosis occurred in RAG−/− mice and depletion of innate lymphoid cells (ILCs) attenuates the fibrotic reaction, suggesting that the pro-fibrotic process is independent of T and B cells. Moreover, IL-22 facilitates the transformation of fibroblasts into myofibroblasts. Finally, the fibrotic reaction was attenuated upon neutralization of either IL-23 or IL-22. Altogether, this study elucidated a signaling cascade underlying intestinal fibrosis in which altered mTOR/autophagy in CX3Cr1+ mononuclear phagocytes up-regulates the IL-23/IL-22 axis, leading to an excessive fibrotic response. Thus, our findings suggest that this cascade could be a therapeutic target for alleviation of CD fibrosis.
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Autophagy is an intracellular catabolic process that is essential for a variety of cellular responses. Due to its role in the maintenance of biological homeostasis in conditions of stress, dysregulation or disruption of autophagy may be linked to human diseases such as inflammatory bowel disease (IBD). IBD is a complicated inflammatory colitis disorder; Crohn’s disease and ulcerative colitis are the principal types. Genetic studies have shown the clinical relevance of several autophagy-related genes (ATGs) in the pathogenesis of IBD. Additionally, recent studies using conditional knockout mice have led to a comprehensive understanding of ATGs that affect intestinal inflammation, Paneth cell abnormality and enteric pathogenic infection during colitis. In this review, we discuss the various ATGs involved in macroautophagy and selective autophagy, including ATG16L1, IRGM, LRRK2, ATG7, p62, optineurin and TFEB in the maintenance of intestinal homeostasis. Although advances have been made regarding the involvement of ATGs in maintaining intestinal homeostasis, determining the precise contribution of autophagy has remained elusive. Recent efforts based on direct targeting of ATGs and autophagy will further facilitate the development of new therapeutic opportunities for IBD.
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Background Colonic subepithelial myofibroblasts (cSEMFs) are mesenchymal cells with a pivotal role in the pathophysiology of Crohn’s disease (CD) fibrosis. Here, we demonstrate for the first time a complete expression mapping of cytokine receptors, implicated in inflammatory bowel diseases, in primary human cSEMFs and how pro-inflammatory cytokines regulate this expression. Furthermore, we show the effect of Th1-, Th2-, Th17- and Treg-related cytokines on a fibrosis-related phenotype of cSEMFs. Methods Colonic subepithelial myofibroblasts were isolated from healthy individuals’ colonic biopsies. Interleukin (IL)-1α- and/or tumor necrosis factor (TNF)–α–induced mRNA and protein expression of cytokine receptors was assayed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence, respectively. Th-related cytokine effects on mRNA and protein profibrotic factor expression were analyzed by qRT-PCR and/or colorimetric assays and on the wound-healing capacity of cSEMFs by scratch test. Results In cSEMFs, we observed basal cytokine receptor expression, which was modified by IL-1α and TNF-α. Th1-related cytokines upregulated tissue factor (TF), collagen, fibronectin and matrix metalloproteinase (MMP)–1 and downregulated α–smooth muscle actin (α-SMA), MMP-9, and wound healing rate. Th2-related cytokines upregulated collagen, TF, α-SMA, MMP-1, and wound healing rate and downregulated fibronectin and MMP-9. IL-17 and IL-23 upregulated fibronectin, and IL-22 downregulated TF. IL-17 and IL-22 decreased wound healing rate. Similar to TGF-β, IL-23 upregulated MMP-1, tissue inhibitor of metalloproteinases–1, collagen expression, and wound healing rates. Conclusions Our results suggest that cSEMFs have a central role in inflammation and fibrosis, as they express a great variety of Th-related cytokine receptors, making them responsive to pro-inflammatory cytokines, abundant in the inflamed mucosa of CD patients.
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Background and pourpose: A defective autophagy is involved in the pathogenesis of inflammatory disorders such as IBD. Cross talk interactions between autophagy and inflammation have been reported and we analyse the effects of autophagy stimulators on murine colitis. Experimental approach: Mice were treated with intrarectal administration of TNBS (3.5 mg/20 g BW) and body weight was measured every day and histological damage score analysed two or four days after treatment. Some mice received trehalose (3% in drinking water three weeks before TNBS administration) or a daily administration of rapamycin (1.25 mg/kg, i.p.), betanin (1g/kg, i.p.) or betanin + 3MA (10mg/kg, i.p.). Mucosal protein levels of p-mTOR, p62, LC3, BCL10, NFκB, IκBα and p-IκBα were determined by WB and mRNA expression of TNFα, IL1β, IL6, IL10, COX2, CCR7, CD11c, iNOS and CD86 by qRT-PCR. Key results: An impaired autophagy associated with body weight loss and intestinal damage was detected in the mucosa of TNBS-treated mice. Administration of trehalose, rapamycin or betanin prevented the impaired autophagic flux induced by TNBS and decreased mucosal protein levels of BCL10, p-IκBα and NFκBp65 and the expression of pro-inflammatory cytokines and M1 macrophage markers. Blockade of the autophagosome formation by treatment of mice with 3MA, prevented the reduction in protein levels of p62, BCL10, p-IκBα and NFκBp65 induced by betanin in TNBS-treated mice and weakened the protective effects of betanin on murine colitis. Conclusions and implications: Our results demonstrate that pharmacological stimulation of mucosal autophagy reduces intestinal inflammation and ameliorates murine colitis.
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A defective induction of epithelial autophagy may have a role in the pathogenesis of inflammatory bowel diseases. This process is regulated mainly by extracellular factors such as nutrients and growth factors and is highly induced by diverse situations of stress. We hypothesized that epithelial autophagy is regulated by the immune response that in turn is modulated by local hypoxia and inflammatory signals present in the inflamed mucosa. Our results reveal that HIF-1α and Wnt1 were co-localized with CD68 in cells of the mucosa of IBD patients. We have observed increased protein levels of β-catenin, phosphorylated mTOR, and p62 and decreased expression of LC3II in colonic epithelial crypts from damaged mucosa in which β-catenin positively correlated with phosphorylated mTOR and negatively correlated with autophagic protein markers. In cultured macrophages, HIF-1 mediated the increase in Wnt1 expression induced by hypoxia, which enhanced protein levels of β-catenin, activated mTOR, and decreased autophagy in epithelial cells in co-culture. Our results demonstrate a HIF-1-dependent induction of Wnt1 in hypoxic macrophages that undermines autophagy in epithelial cells and suggest a role for Wnt signaling and mTOR pathways in the impaired epithelial autophagy observed in the mucosa of IBD patients.Mucosal Immunology advance online publication, 4 December 2013; doi:10.1038/mi.2013.108.
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Purpose of review: Intestinal fibrosis is a serious, yet common, outcome in patients with inflammatory bowel disease (IBD). Despite advances in developing novel treatment modalities to control chronic gut inflammation characteristic of IBD, no effective antifibrotic therapies exist to date. As such, a deeper understanding of the molecular mechanisms underlying intestinal fibrosis and the availability of relevant animal models are critical to move this area of investigation forward. Recent findings: Emerging concepts in the pathogenesis of intestinal fibrosis include the central role of interleukin (IL)-17 and Th17 immune responses, although their precise contribution to chronic inflammation and IBD remains controversial. Other novel mediators of intestinal fibrosis, such as tumor necrosis factor-like ligand 1A and components of the renin-angiotensin system, support the importance of IL-17. Additionally, recent studies utilizing novel mouse models highlight the significance of the gut microbiota and link components of bacterial sensing, including nucleotide-binding oligomerization domain-containing protein 2, to IL-17/Th17 immune responses in the development of inflammation-associated intestinal fibrosis. Summary: Recent progress in identifying key mediators, novel animal models, and important mechanistic pathways in the pathogenesis of intestinal fibrosis holds promise for the development of effective antifibrotics in an area of significant, unmet clinical need.
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Background and aims Little evidence demonstrating the correlation between several single nucleotide polymorphisms and a specific phenotype of Crohn's disease has been reported in children. We investigated the relationship between autophagy genes variants and clinical features in our children with Crohn's disease. Methods Genotyping for ATG16L1, NOD2/CARD15, and IRGM1 was performed in 80 consecutive patients with Crohn's disease (median age: 11 years; range: 0.7–17.9 years). Crohn's disease location and behaviour were classified using the Paris classification. Additional data were collected from clinical records on patients’ demographics, age at symptom onset and diagnosis, extraintestinal manifestations, therapy, clinical relapses, and need of surgical intervention. Results Patients homozygous for the risk allele ATG16L1 (T300A) showed a trend towards switching to a stricturing phenotype during the course of disease compared to children either homozygous for the wild-type allele or heterozygous for the ATG16L1 single nucleotide polymorphism (p = 0.01). Homozygosity for the ATG16L1 risk allele was associated with a major recurrence of clinical relapses and earlier introduction of immunosuppressants (p = 0.006 and p = 0.04, respectively). Heterozygosity for the NOD2 rs2066847 allele was associated with major ileal involvement (p = 0.01). Conclusion In patients carrying the T300A variant, Crohn's disease follows a more aggressive clinical course.