Autophagy Stimulation as a Potential Strategy Against Intestinal Fibrosis

Abstract

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.

Full text

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 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.
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 effects have been attributed to
autophagy stimulation in different organic systems [
4
,
5
]. Hence, we aim to analyze the effects 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
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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 paraffin-embedded colonic tissues in order
to analyze the collagen layer in intestinal grafts obtained after the heterotopic transplant model. After
deparaffinization 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 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 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 differences vs. control intestinal tissues are shown by * p<0.05,
** p<0.01 and
*** p<0.001
; significant differences 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 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].

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 (5 ng/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 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).
4. Discussion
This study demonstrates a reduced autophagy in murine intestinal fibrosis. Autophagy stimulation
exerts an anti-fibrotic effect 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 different 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 effect 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 affected
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 effect of autophagy
stimulation and the consequent inhibition of the IL-22/IL-23 axis in macrophages has been recently
demonstrated in a different 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 affecting its gene expression. This
implies that fibroblast’s autophagy, and its pharmacological regulation, affects collagen degradation.
This mechanism most probably contributes to the effects 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.
References
1.
Kim, S.; Eun, H.S.; Jo, E.K. Roles of Autophagy-Related Genes in the Pathogenesis of Inflammatory Bowel
Disease. Cells 2019,8, 77. [CrossRef] [PubMed]
2.
Ortiz-Masia, D.; Cosin-Roger, J.; Calatayud, S.; Hernandez, C.; Alos, R.; Hinojosa, J.; Apostolova, N.;
Alvarez, A.; Barrachina, M.D. Hypoxic macrophages impair autophagy in epithelial cells through Wnt1:
Relevance in IBD. Muc. Immunol. 2014,7, 929–938. [CrossRef] [PubMed]
3.
Macias-Ceja, D.C.; Cosin-Roger, J.; Ortiz-Masia, D.; Salvador, P.; Hernandez, C.; Esplugues, J.V.; Calatayud, S.;
Barrachina, M.D. Stimulation of autophagy prevents intestinal mucosal inflammation and ameliorates
murine colitis. Br. J. Pharmacol. 2017,174, 2501–2511. [CrossRef] [PubMed]
4.
Del Principe, D.; Lista, P.; Malorni, W.; Giammarioli, A.M. Fibroblast autophagy in fibrotic disorders. J. Pathol.
2013,229, 208–220. [CrossRef] [PubMed]
5.
Hilscher, M.; Hernandez-Gea, V.; Friedman, S.L. Autophagy and mesenchymal cell fibrogenesis. Biochim.
Biophys. Acta 2012,1831, 972–978. [CrossRef] [PubMed]
6.
Macias-Ceja, D.C.; Ortiz-Masia, D.; Salvador, P.; Gisbert-Ferrandiz, L.; Hernandez, C.; Hausmann, M.;
Rogler, G.; Esplugues, J.V.; Hinojosa, J.; Alos, R.; et al. Succinate receptor mediates intestinal inflammation
and fibrosis. Mucosal. Immunol. 2019,12, 178–187. [CrossRef] [PubMed]

Cells 2019,8, 1078 8 of 8
7.
Babicki, S.; Arndt, D.; Marcu, A.; Liang, Y.; Grant, J.R.; Maciejewski, A.; Wishart, D.S. Heatmapper:
Web-enabled heat mapping for all. Nucleic Acids Res. 2016,44, W147–W153. [CrossRef] [PubMed]
8.
Fowler, E.V.; Doecke, J.; Simms, L.A.; Zhao, Z.Z.; Webb, P.M.; Hayward, N.K.; Whiteman, D.C.; Florin, T.H.;
Montgomery, G.W.; Cavanaugh, J.A.; et al. ATG16L1 T300A shows strong associations with disease
subgroups in a large Australian IBD population: further support for significant disease heterogeneity. Am. J.
Gastroenterol. 2008,103, 2519–2526. [CrossRef] [PubMed]
9.
Strisciuglio, C.; Auricchio, R.; Martinelli, M.; Staiano, A.; Giugliano, F.P.; Andreozzi, M.; De Rosa, M.;
Giannetti, E.; Gianfrani, C.; Izzo, P.; et al. Autophagy genes variants and paediatric Crohn’s disease
phenotype: A single-centre experience. Dig. Liver. Dis. 2014,46, 512–517. [CrossRef] [PubMed]
10.
Salvador, P.; Macias-Ceja, D.C.; Gisbert-Ferrandiz, L.; Hernandez, C.; Bernardo, D.; Alos, R.;
Navarro-Vicente, F.; Esplugues, J.V.; Ortiz-Masia, D.; Barrachina, M.D.; et al. CD16+Macrophages Mediate
Fibrosis in Inflammatory Bowel Disease. J. Crohns Colitis 2018,12, 589–599. [CrossRef]
11.
Chang, J.; Hisamatsu, T.; Shimamura, K.; Yoneno, K.; Adachi, M.; Naruse, H.; Igarashi, T.; Higuchi, H.;
Matsuoka, K.; Kitazume, M.T.; et al. Activated hepatic stellate cells mediate the differentiation of macrophages.
Hepatol. Res. 2013,43, 658–669. [CrossRef] [PubMed]
12.
Liaskou, E.; Zimmermann, H.W.; Li, K.K.; Oo, Y.H.; Suresh, S.; Stamataki, Z.; Qureshi, O.; Lalor, P.F.;
Shaw, J.; Syn, W.K.; et al. Monocyte subsets in human liver disease show distinct phenotypic and functional
characteristics. Hepatology 2013,57, 385–398. [CrossRef] [PubMed]
13.
Ray, S.; De Salvo, C.; Pizarro, T.T. Central role of IL-17/Th17 immune responses and the gut microbiota in the
pathogenesis of intestinal fibrosis. Curr. Opin. Gastroenterol. 2014,30, 531–538. [CrossRef] [PubMed]
14.
Filidou, E.; Valatas, V.; Drygiannakis, I.; Arvanitidis, K.; Vradelis, S.; Kouklakis, G.; Kolios, G.; Bamias, G.
Cytokine Receptor Profiling in Human Colonic Subepithelial Myofibroblasts: A Differential Effect of Th
Polarization-Associated Cytokines in Intestinal Fibrosis. Inflamm. Bowel. Dis.
2018
,24, 2224–2241. [CrossRef]
[PubMed]
15.
Mathur, R.; Alam, M.M.; Zhao, X.F.; Liao, Y.; Shen, J.; Morgan, S.; Huang, T.; Lee, H.; Lee, E.; Huang, Y.
Induction of autophagy in Cx3cr1(+) mononuclear cells limits IL-23/IL-22 axis-mediated intestinal fibrosis.
Mucosal. Immunol. 2019,12, 612–623. [CrossRef] [PubMed]
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