microRNA-155 inhibition restores Fibroblast Growth Factor 7 expression in diabetic skin and decreases wound inflammation
microRNA-155 inhibition restores Fibroblast Growth Factor 7 expression in diabetic skin and decreases wound inflammation
Jo?o Moura 0
Anja S?rensen 1
Ermelindo C. Leal 0
Rikke Svendsen 1
Lina Carvalho 2
Rie Juul Willemoes 1
Per Trolle J?rgensen Louise Torp Dalgaard
0 c enter for n euroscience and c ell Biology, University of c oimbra , c oimbra , Portugal
1 Department of Science and environment, Roskilde University , Roskilde , Denmark
2 f aculty of Medicine, University of c oimbra , c oimbra , Portugal
3 nucleic Acid center, Department of Physics , chemistry and Pharmacy , University of Southern Denmark , Odense , Denmark
4 Department of Geriatrics, University of Arkansas for Medical Sciences , Little Rock, Arkansas , United States
5 Arkansas children's Research i nstitute , Little Rock, Arkansas , United States. Joa?o Moura, Anja S?rensen
Treatment for chronic diabetic foot ulcers is limited by the inability to simultaneously address the excessive inflammation and impaired re-epithelization and remodeling. Impaired re-epithelization leads to significantly delayed wound closure and excessive inflammation causes tissue destruction, both enhancing wound pathogen colonization. Among many differentially expressed microRNAs, miR-155 is significantly upregulated and fibroblast growth factor 7 (FGF7) mRNA (target of miR-155) and protein are suppressed in diabetic skin, when compared to controls, leading us to hypothesize that topical miR-155 inhibition would improve diabetic wound healing by restoring FGF7 expression. In vitro inhibition of miR-155 increased human keratinocyte scratch closure and topical inhibition of miR-155 in vivo in wounds increased murine FGF7 protein expression and significantly enhanced diabetic wound healing. Moreover, we show that miR-155 inhibition leads to a reduction in wound inflammation, in accordance with known pro-inflammatory actions of miR-155. Our results demonstrate, for the first time, that topical miR-155 inhibition increases diabetic wound fibroblast growth factor 7 expression in diabetic wounds, which, in turn, increases re-epithelization and, consequently, accelerates wound closure. Topical miR-155 inhibition targets both excessive inflammation and impaired re-epithelization and remodeling, being a potentially new and effective treatment for chronic diabetic foot ulcers.
Chronic diabetic foot ulceration (DFU) is one of the most debilitating complications of long-standing diabetes.
DFU is, at least in part, a consequence of uncontrolled infection of foot wounds, due to the presence of
neuropathy, peripheral vascular/arterial disease1, impaired angiogenesis and chronic low-grade inflammation2. Reduced
blood flow restricts migration of leukocytes3, keratinocytes, fibroblasts and endothelial progenitor cells to the
wounded site4. Long-term hyperglycemia promotes the activation of NFkB5, leading to chronic inflammation2
and impairing leukocyte activation and migration3. Despite the huge impact of long-term hyperglycemia on
the progression of diabetes complications, the ACCORD and ADVANCE clinical trials6 showed that while
glucose-lowering treatments reduce the risk of cardiovascular diseases, the risk of DFU and other complications
still remains, thus, indicating that current therapies are not sufficient and there is an urgent need to identify better
Several microRNAs (miRs) have been associated with DFU progression and severity7. While specific miRs,
such as miR-318, have been shown to improve wound healing, other miRs, such as miR-26a9, increase the
severity of DFU. miR-155 fits in this second category10?13. It is a small (23 nucleotide), single-stranded, non-coding
RNA originally identified as a gene on human chromosome 21, formerly called B-cell Integration Cluster14. The
pre-miR-155 hairpin gives rise to two mature forms (miR-155-3p and miR-155-5p)15, where miR-155-5p is the
(miRbase MI0000681, from now on referred as miR-155)
In murine models, whole-body over-expression of miR-155 leads to hypoglycemia, because miR-155
positively regulates insulin sensitivity and glucose uptake in insulin-sensitive cells, whereas complete deficiency of
miR-155 results in hyperglycemia11. MiR-155 is expressed by immune cells16, including Th1 and Th1717, as well
as other cells in inflammatory conditions18, and plays a pro-inflammatory role in cells, by targeting Cytotoxic
T-Lymphocyte-associated protein (CTLA)- 419, suppressor of cytokine signaling (SOCS)1, and SH2-Containing
Inositol-5?-phosphatase (SHIP)1 from the Toll-Like Receptor (TLR)- 2 pathway20. Furthermore, inhibition of
regulatory T cells by miR-155, both in control17 and diabetic subjects13, promotes the exacerbation of
inflammation, which is involved in the pathology of psoriasis17. Moreover, anti-inflammatory drugs such as L-arginine and
ibuprofen12, resveratrol21, vitamin D22 or M200023 have been shown to downregulate miR-155.
MiR-155 is also important for the function of skin cells involved in wound healing, including keratinocytes24,
dermal mesenchymal stem cells25, mast cells26, melanocytes27, adipocytes28 and fibroblasts10. Furthermore,
miR155 deficiency29 and miR-155 inhibition30 was shown to improve wound healing, in healthy and diabetic animal
models, but the mechanism by which miR-155 impairs wound healing remains elusive.
In this work, we aimed to investigate the effect of specifically inhibiting miR-155 in diabetic skin on wound
healing in a type 1 diabetic mouse model. Our diabetic mouse model confirmed a significant increase in
miR155 expression in the skin during wound healing and topical miR-155 inhibition improved wound closure via
de-repression of FGF7 (fibroblast growth factor 7).
Wound healing is compromised under diabetic conditions, in humans3 and in rodents31. To confirm wound
healing impairment, we used a well-established mouse model of diabetic wound healing31. Wound closure was
monitored for 10 days post-wounding. Our results demonstrate wound healing impairment in diabetic mice (Fig.?1A).
To evaluate the effect of diabetes on skin miR expression during wound healing, we used skin samples collected
at baseline (Day 0) and Days 3 and 10 post-wounding and profiled miR expression. The array screening results
indicate that in diabetic mice (n = 6 in each group ? days 0, 3 and 10), a large fraction of the detected miRs show
more than two-fold difference in expression at days 0, 3 or 10 following wounding (Fig.?1B): 36, 29 and 17 miRs
were upregulated and 44, 35 and 37 miRs were downregulated in diabetic skin at these time points.
Array results were further validated by RT-qPCR (reverse transcription quantitative polymerase chain
reaction) for individual targets (Fig.?2). Interestingly, while miR-155 was significantly overexpressed in diabetic
mouse skin, compared to non-diabetic mice (15.8 fold ? 4.8 vs 1.0 ? 0.4, p < 0.001) at baseline, it was
markedly decreased in diabetic skin (2.3 ? 0.5 vs 22.3 ? 5.8, p < 0.05) in the later phases of wound healing (Day 10).
Similar to miR-155, miR-126-5p was increased in diabetic skin at Day 0 and suppressed at Day 10 compared
with non-diabetic skin. A number of other miRs were significantly decreased in diabetic skin wounds at Day 10:
miR-17-5p, miR-31-3p, miR-31-5p, miR-324-3p and miR-411-5p, while miR-127-3p was downregulated at Day 3
and 10 in diabetic wounded skin. miR-21-5p was increased at Day 0 in diabetic skin, but not at other time points.
Since a large fraction of the analyzed miRs were decreased in diabetic mouse skin, we further evaluated the
mRNA expression levels of four proteins involved in miR processing, TAR RNA Binding Protein 1 (Trbp-1),
DiGeorge Syndrome Chromosomal Region 8 (Dgcr8), Dicer and Drosha-2 (Fig.?3). Interestingly, mRNA levels
of Drosha-2, Dgcr8 and Dicer were significantly increased (p < 0.001 for Drosha-2 and Dgcr8 and p < 0.01 for
Dicer) in diabetic mouse skin, before and after wound induction, when compared to non-diabetic mice. While
the overall miR expression was decreased at Day 0 in diabetic mice, the increased RNA transcript level of these
factors involved in miR processing (Drosha-2, Dgcr8, Dicer, Trbp1) may suggest an unaccommodated feedback
from decreased miR action in diabetic mouse skin at Day 0.
Since miR-155 was one of the most differentially expressed miRs in diabetic and control skin and it has
previously been shown to be important for wound healing29, we chose to investigate miR-155 in depth. Importantly,
we also evaluated the expression of one in silico identified target of miR-155?s, FGF7 (also known as keratinocyte
growth factor) (Fig.?4A). As opposed to control mice, where FGF7 mRNA levels decrease during wound healing,
in diabetic mouse skin, the levels of FGF7 mRNA inversely correlate with the expression of miR-155, increasing
significantly during wound healing. To test miR-155 efficacy in downregulating FGF7 expression, we constructed
an FGF7-luciferase-reporter vector containing the 3? UTR (untranslated region) of the FGF7 gene ((FGF7 UTR)
(Fig.?4B) and analyzed luciferase activity in response to different concentrations of miR-155 inhibitor in human
HaCaT keratinocytes. FGF7 UTR mediated reporter-gene activity increased (FGF7 UTR: 1.65-fold ? 0.06 at
25 pmol inhibitor/well and 1.99-fold ? 0.12 at 35 pmol inhibitor/well compared with control, both p < 0.001)
with miR-155 inhibition in a dose-dependent manner. Importantly, removing just one of the predicted two
miR155 target sites of the FGF7 UTR significantly decreases the response to miR-155 inhibition (Fig.?4B).
Furthermore, we performed an in vitro scratch-assay to test the effect of miR-155 on cell migration.
Transfection of HaCaT keratinocyte cells with full-length miR-155 inhibitor significantly increased in vitro
scratch closure, in hyperglycemic conditions (Fig.?4C): While negative control transfected cells had 31.4% ? 3.9%
remaining scratch after 24 hrs, miR-155 inhibitor transfected cells had only 8.1% ? 2.9% remaining (p < 0.0001).
Moreover, scratch closure was also significantly enhanced when using a shorter inhibitor only binding to the seed
site of miR-155 (Seed 155 inhibitor) (Seed Neg. Ctrl.: 43.1% ? 5.0% vs Seed 155 inhibitor:18.2% ? 5.2%) (Fig.?4C
and Suppl. Fig.?1).
While the scratch wound in vitro assay is a simple wound healing model and does not reflect the multiple
cellular interactions taking place in in vivo, we performed wound healing experiments in diabetic mice. To test
the in vivo effect of miR-155 inhibition we measured wound closure kinetics over a period of 10 days (Fig.?5A).
Dorsal wounds were induced on the back of diabetic mice and were subsequently topically treated with different
concentrations of the miR-155 inhibitor, twice a day, until Day 3 (n = 6 in each group). Topical administration of
the miR-155 inhibitor after wound induction significantly improved wound closure, especially with 2.5 nmol dose
applied. Improvement in wound closure was visible as early as the first day of treatment and was persistent even
after the 3 days of treatment until the end of the experiment, 10 days post-wounding. The 0.25nmol dose had
no effect on wound healing and 2.5 nmol and 10 nmol doses had a similar effect (data not shown). Moreover, the
negative control inhibitor did not display altered wound healing kinetics compared with the saline control (data
not shown), showing that the action of the miR-155 inhibitor is very likely to be sequence specific.
HE and Herovici?s staining at Day 10 post-wounding indicated anti-inflammatory effects of in vivo miR-155
inhibition (Fig.?5B). As opposed to diabetic mouse wounds treated with the negative inhibitor, where a clear
immune cell infiltration was observed, diabetic mouse wounds treated with 2.5 nmol of miR-155 inhibitor
presented a complete re-epithelization, with no traces of inflammation. We also observed a prevalence of young,
unorganized collagen in control wounds (Fig.?5B), in clear contrast with miR-155 inhibitor treated wounds, where
young collagen expression was residual, indicating that miR-155 inhibition accelerates wound maturation. To
further evaluate immune cell infiltration at Day 10 post-wounding, we performed fluorescent immunohistochemical
staining for CD3, to identify T-cells and CD68 to identify macrophages (Fig.?6) on control and miR-155 inhibitor
treated wounds (n = 3 in each group). Macrophage wound infiltration was significantly decreased (32% of Ctrl.)
in diabetic wounds treated with 2.5 nmol of miR-155 inhibitor in control diabetic wounds than (Ctrl.: 77.7 ? 3.1
vs MiR-155 inhibitor: 24.7 ? 1.8 cells/field; p < 0.0001) (Fig.?6B), while T-cell infiltration was 39% of Ctrl.
following inhibition of miR-155 (Ctrl.: 22.0 ? 6.0 vs MiR-155 inhibitor: 8.7 ? 1.1 cells/field, p = 0.05) (Fig.?6C).
To elucidate the action of the miR-155 inhibitor on FGF7 expression, we performed fluorescent
immunohistochemical staining for FGF7 (Fig.?7A), on samples collected at Day 10 post-wounding. We stained non-diabetic
wounded skin and observed high levels of FGF7 associated with hair shafts, as well as staining throughout sub
cutis and the epidermis. In diabetic skin, treated with the negative control inhibitor oligo, FGF7 levels were clearly
suppressed (3.8% ? 2.5%, p < 0.001 compared to non-diabetic control), both in the hair shaft follicles and in the
epidermis. Treatment with miR-155 inhibitor markedly increased FGF7 levels in hair shafts, in the subcutis and
in the epidermis of diabetic mice (41.3% ? 19.6%, p < 0.01 compared to diabetic mice)(Fig.?7B). Thus, FGF7 is
clearly de-repressed at the protein level by miR-155 inhibition, while the shorter Seed miR-155 inhibitor had no
effect in vivo, under the tested conditions.
We tested the action of the miR-155 inhibitor on FGF7 mRNA levels in skin from diabetic mice wounded and
subsequently topically treated with inhibitor. Despite clear functional actions of the miR-155 inhibitor at Day 3,
there was no effect of the miR-155 inhibitor on steady state FGF7 mRNA level or on other predicted mRNA
targets (Ctla4, Hbp1, Stat3, Nfia) (data not shown). Similarly, despite clear effects on scratch-assay migration (Suppl.
Fig.?1), the miR-155 inhibitor did not alter measured miR-155 levels or FGF7 mRNA levels (data not shown). We
also evaluated the effect of miR-155 inhibitor on angiogenesis, by staining the vessels for CD31, and we did not
observe changes between the negative control oligo and the miR-155 inhibitor (Suppl. Fig.?2).
Skin injury triggers acute inflammatory responses, beginning with recruitment of neutrophils and monocytes
to the site of injury that, in turn, secrete various inflammatory cytokines, chemokines and growth factors
coordinating wound repair32. Excessive inflammation observed in DFU causes tissue damage through the release of
increasing levels of various proteins involved in bacterial control, such as granzymes and perforins, that degrade
the extracellular matrix33 and inhibit re-epithelization34 impairing wound healing. This excessive inflammation is
partially controlled by miRs, including miR-15535, and its suppression, either directly using a miR-155 inhibitor36
or indirectly using other substances21,37, has an anti-inflammatory effect in different conditions.
Our results show that miR-155 is over-expressed in diabetic mouse skin, when compared to non-diabetic
control mice. Most importantly, we show, for the first time, that FGF7 mRNA expression is significantly decreased
studies have shown that FGF7 over-expression favors wound healing in various ways that are not restricted to
keratinocyte or fibroblast proliferation and migration, but also involve increased revascularization and
antimicrobial effects46. Here, we demonstrate that topical inhibition of miR-155 leads to increased keratinocyte migration
and faster wound closure, in a dose-dependent manner. We also show in vitro that miR-155 regulates keratinocyte
migration through inhibition of the FGF7 3? untranslated region (UTR).
However, when evaluating the temporal modulation of miR-155 and FGF7 it is apparent that other factors
than miR-155 are likely to contribute to the regulation of FGF7 mRNA and protein levels. For example, in diabetic
animals miR-155 peaks at day 0 and is significantly less expressed at day 10, while no difference in FGF7 mRNA
abundance is observed at day 0 between diabetics and controls, and FGF7 transcript is significantly more
abundant at day 10 in diabetics. When considering other miRNAs as regulators then miR-21 also has a target site in
the FGF7 3?UTR, which may contribute to suppression of FGF7 protein at day 0 in diabetic skin (where miR-21 is
upregulated). Moreover, other factors than microRNAs (such as inflammatory cytokines47) control FGF7 mRNA
and protein amounts. In addition, inhibition by microRNAs on their mRNA or protein targets is not
instantaneous but rather slow, and the temporal effect of a microRNA also depends on the turnover of the protein, providing
a possible explanation for discrepant observations between the miR-155 and FGF7 levels.
The FGF7 mRNA levels and FGF7 immunostainings in diabetic skin at day 10 following wounding did not
correlate, but the mRNA of FGF7 in diabetic skin could be increased in a compensatory response. Not all
microRNAs act to cause complete degradation of their cognate mRNA targets, but instead primarily halts the translation
of the mRNA into protein. In fact, we could not detect significant increase in the FGF7 mRNA by treatment with
the miR-155 inhibitor (data not shown), despite clear increases at the FGF7 protein levels and responses of the
FGF7 3?UTR to miR-155 inhibitor in reporter assays.
Our results suggest that topical administration of miR-155 inhibitors, at the wound site, may have significant
therapeutic value in DFU treatment, especially if applied during the first days after wounding, where miR-155
inhibition will have an immunosuppressive effect and decrease tissue damage.
Multiple low-dose streptozotocin diabetic mouse model. C57BL/6 male mice (25?30 g) obtained
from Charles River Corporation Inc. (Barcelona, Spain) were housed at 22?24 ?C with a 12 hrs light/dark
cycle with ad libitum access to water and food. Experimental protocols were in accordance with the European
Community law for Experimental Animal studies (86/609/CEE; 2007/526/CE) and were approved by the
Institutional (Organ Responsible for Animal Welfare of the Center for Neurosciences and Cell Biology and
Faculty of Medicine of the University of Coimbra) and Governmental (Directorate-General for Food and
Veterinary of the Portuguese Ministry of Agriculture) Research Ethical Boards.
Diabetes was induced as previously described31. Briefly, streptozotocin (STZ) (50 mg/kg) in saline solution
was injected i.p., for 5 consecutive days. Seven days post STZ injection, blood glucose was measured to confirm
the diabetic phenotype. Mice with blood glucose levels above 250 mg/dL (Accu-Chek glucometer, Roche, Basel/
Switzerland), were considered diabetic. Animals were treated with isophane (NPH) insulin (0.1?0.2 units),
subcutaneously, as needed, to avoid weight loss. Animals were kept diabetic for 6 weeks prior to the wounding
experiments. Like diabetic patients, STZ-induced diabetic mice also develop chronic low-grade inflammation leading
to wound healing impairment31.
Wound healing model and treatments. Wound induction was performed as previously described31.
Briefly, control or diabetic mice were anesthetized with Ketamine/Xylazine (100/10 mg/kg, i.p.). After
removing the dorsal hair, two 6 mm excisional wounds 2 cm apart were created using a punch biopsy tool (Miltex,
Rietheim-Weilheim, Germany). The wound area was traced daily onto acetate paper to follow rates of wound
closure for 10 days post-wounding. Wound size was determined with ImageJ version 1.46 (NIH Image, USA).
Diabetic mice were used for miR-inhibitor treatments. Both wounds were treated topically, twice daily up to
day 3 post wounding, with a miR-155 inhibitor (5?-TcaCaaTuaGcaTuaA-3?) (0.25, 1, 2.5 or 10 nmol) or with a
negative control oligo (5?-CaaTagGguCaaGauT-3?); locked nucleic acid (LNA) bases are written in capital letters
while 2?-O-methyl RNA bases in small letters. Oligos were designed using LNA bases for every third nucleotide
and the backbone was phosphorothioate substituted for enhanced binding strength and stability. A seed miR-155
inhibitor (5?-AGCAuTaA-3?) was synthesized to only target the seed sequence of miR-155, and a seed control
oligo (5?-TCAAgAuT-3?) was also synthesized48. Animals were sacrificed at days 3 or 10 post-wounding and the
wounded skin was harvested for analysis.
Skin wound homogenization and RNA extraction. Skin tissue (50?100 mg) was homogenized with
1 mL TRI Reagent (Sigma Aldrich, St. Louis, Missouri, USA) with a polytron homogenizer followed by
purification as per manufacturers? instructions. The RNA pellet was dissolved in DEPC water (50? L). RNA concentration
and purity were assessed using the NanoDrop ND-1000 spectrophotometer (ThermoFisher Scientific, Waltham,
Massachusetts, USA). Samples were stored at ?80 ?C until further analysis.
Profiling miRs in skin wounds. MiR expression was measured on pools of skin samples using Rodent
TaqMan Low Density Array cards, v.2.0 for RT-primer pool A and v3.0 for RT-primer pool B, containing 641
unique murine miRs (ThermoFisher Scientific, Waltham, Massachusetts, USA) according to
manufacturer?s instructions. Each pool contained six samples from the same experimental group, with total RNA input of
600 ng per array card. Briefly, total RNA was reverse-transcribed using multiplex RT primer pool sets followed by
quantitative PCR (qPCR) step with sequence-specific primers and probes on the TaqMan? MicroRNA Arrays.
Expression data were obtained using the Viia 7 qPCR system (ThermoFisher Scientific, Waltham, Massachusetts,
USA). Data was normalized against the stably expressed U6 snRNA available on the array and relative miR
expression was calculated using the comparative ??Ct method (2???Ct), against the expression of the same miR
in control mice at day 0.
Measurement of miR and mRNA levels by RT-qPCR. miRs were detected using reverse-transcription
quantitative PCR (RT-qPCR) as previously described49. Oligonucleotides used for reverse transcription and
qPCR are shown in Supplementary Table?1. Fibroblast growth factor (FGF) 7 mRNA levels were quantified using
qPCR primers (QT00172004, Qiagen, Hilden/Germany), with Quantitect Sybr 2x Master Mix (Qiagen, Hilden,
Germany) in 10 ? L reactions using the MX3005 qPCR system (Agilent, Santa Clara, California, USA). Transcripts
were quantified using standard curve quantification, diluted skin cDNA served as input to generate the standard
curve. The geometric mean of transcription factor (TF)IIB and U6 levels were used to normalize for variation in
input template. The geometric mean of these two transcripts was unaltered.
Cell culture and transfections. Human clonal keratinocyte (HaCaT) cells were cultured in Dulbeccos
Modified Eagle Medium (DMEM, 20 mM glucose) (ThermoFisher Scientific, Waltham, Massachusetts, USA)
supplemented with FBS (10%) and Penicillin/Streptomycin (1%). MiR-155 levels were measured by RT-qPCR
and this microRNA is expressed in HaCaT cells although at moderate levels, with Ct levels from 1 ng cDNA
starting at cycle 25?27. For scratch migration assays, HaCaT cells were seeded 30.000 cells per well in 48 well plates
and allowed to adhere for 24 hrs, before transfection with miR inhibitors. Transfections consisted of inhibitor
(25 pmol) and Lipofectamine 2000 (0.5 ? L) (ThermoFisher Scientific, Waltham, Massachusetts, USA) per well
and were prepared according to manufacturer instructions in triplicate. After 24 hrs the medium was changed
to DMEM containing glucose (20 mM) supplemented with FBS (10%) and Penicillin/Streptomycin (1%) and
scratches were performed. Following washes in medium to remove non-adherent cells, microscope images were
acquired at time zero and 24 hrs later. Distance between scratch edges was calculated using ImageJ and are
presented as percentage of remaining scratch after 24 hrs.
Wild type and mutant FGF7 3? untranslated region (UTR) constructs for miR-155-5p site 1 were cloned by
PCR amplification of mouse genomic DNA (oligonucleotides listed in Tab. 1) and inserted into the XbaI and FseI
sites in the 3? UTR of the luc2 gene of pGL4.13 (Promega, Madison, Wisconsin, USA).
For reporter-gene analysis of 3? UTR reporter constructs, HaCaT cells were seeded in 48 well plates (20.000
cells per well) and allowed to adhere for 24 hrs. Following media change, transfections consisting of 35 ng beta-gal
expression plasmid and 315 ng luciferase UTR reporter-gene plasmid with or without miR inhibitor (amounts
indicated in figures) were made using linear polyethylineimine (PEI25) (Sigma Aldrich, St. Louis, Missouri,
USA)50. After 24 hrs cells were lysed and luciferase measured using the DualLight Assay (Perkin Elmer, Waltham,
Massachusetts, USA) on a GloMax96 instrument with a dual-injector system (Promega, Madison, Wisconsin,
USA). Transfections were made in triplicate and luciferase activity was normalized to beta galactosidase activity
to control for differences in cell transfection rate.
Immunofluorescence staining and histological analysis. Samples from frozen optimal cutting tem
perature compound (OCT) blocks were sectioned with a 10 ? m thickness, put on glass slides and kept at ?20 ?C
for later use. Thawed skin sections were incubated with anti-FGF7 (1:100) (PA5-49715, Invitrogen, Carlsbad,
California, USA), anti-CD3 (1:100) (PC630, Merck Millipore, Darmstadt, Germany), anti-CD68 (1:100) (ab955,
Abcam, Cambridge, UK) or anti-CD31 (1:200)(Merck Millipore, Darmstadt, Germany) and the nuclei were
stained with DAPI (Sigma Aldrich, St. Louis, Missouri, USA). After washing with PBS, sections were incubated
with Alexa fluor 488 conjugated goat antiserum against rabbit (1:250) (Invitrogen, Carlsbad, California, USA) or
Alexa fluor 594 conjugated goat antiserum against rat (1:500). The sections were then imaged using a confocal
(FGF7, CD3 and CD68) or fluorescence (CD31) microscope. FGF7 was quantified by measuring the
fluorescence intensity of two independent microscopy fields with 200x magnification and T-cells (CD3+), macrophages
(CD68+). The CD31 was measured by counting the number of vessels from ten images of three different
sections (200x magnification). Hematoxylin and Eosin (HE) (Merck Millipore, Darmstadt, Germany) and Herovici?s
(American Mastertech Scientific, Lodi, California, USA) stainings were performed in 3 ? m thickness paraffin
sections, according to the manufacturer?s instructions. The sections were imaged using a transmission microscope.
Statistics. GraphPad Prism and Excel were used for statistical analysis. Significance was tested by Students
t-test (for 2 groups), repetitive, paired t-test (in vivo wound healing analysis) or one or two-way ANOVA with
Dunnets? post hoc correction (multiple groups) with a significance level of p < 0.05. Data shown are averages
from replica experiments.
Competing Interests: The authors declare no competing interests. Publisher?s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. The images or other third party material in this
article are included in the article?s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article?s Creative Commons license and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
JM acknowledges a postdoctoral fellowship from the HealthyAging2020 project , CENTRO-01-0145-FEDER000012-N2323 . ECL acknowledges a postdoctoral fellowship from the Portuguese Foundation for Science and Technology , SFRH/BPD/112883/ 2015 . AES acknowledges a post doctoral fellowship from the Danish Diabetes Academy, supported by the Novo Nordisk Foundation. The authors would like to thank Christa Persson for dedicated and skilled technical expertise. This work was supported by the European Foundation for the Study of Diabetes to LTD, HJ and EC, the Danish Medical Research Council to LTD , GIFT/SPD to EC, HealthyAging2020 CENTRO- 01 -0145-FEDER-000012 -N2323 to EC, as well as Pepper grant: P30 AG028718 and NIGMS_NIH P20GM109096 .
Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-42309-4.