Patching up the crypt - RePub, Erasmus University Repository

Patching up the cryptInnate immune cells orchestrate

intestinal regeneration

Mónica Romera Hernández

Patching up the crypt: Innate immune cells orchestrate

....Copyright © 2018

All rights reserved.

in any form or by any means without permission from the author. The copyright of

Layout: Egied Simons Cover: Iliana Boshoven-Gkini

The work described in this thesis was performed at the Department of Hematology at the

the Netherlands.

Patching up the crypt:

Innate immune cells orchestrate

aangeboren afweer systeem

op gezag van de

Prof.dr. H.A.P. Pols

De openbare verdediging zal plaatsvinden op

3 mei 2018 om 11:30 uur

door

Mónica Romera Hernández

DOCTORAL COMMITEE

Supervisor: Prof.dr. J.J. Cornelissen

Other members: Prof.dr. I.P. Touw

Prof.dr. F. Koning

Co-supervisor: Dr. T. Cupedo

A mi iaio, de extrema honra y dura proeza

CONTENTS

Chapter 1: Aims and outline of this thesis 26

of Group 3 innate lymphoid cells

English summary

1

GENERAL INTRODUCTION

HARNESSING THE IMMUNE SYSTEM TO ENHANCE INTESTINAL BARRIER FUNCTION

Based in part on: Damage control: Ror

ABSTRACT

1

epithelium by local stem cells, that are contained within a niche in the crypts of Lieberkühn.

11

INTRODUCTION

2,3. Key physical components

self-contained enteric nervous system.

as well as interspersed between epithelial cells, and these contain cell types belonging to

A dynamic balance between the immune system and the epithelium is vital to ensure

4–6.

environment.

.

H , yet

1

12

The family of ILCs can be subdivided in three main types based on their master

+ + ILC3 subtypes

11,12

ILCs depend on c13,14 but in

-lymphoid progenitors ( + integrin 4+-

4+

Id2hi

and ILCP progenitors are able to generate ILC1, ILC2 and NKp46+ + ILC3s . Id2+ -

4+ ILC3s12,20. The

21

is not known22–24

.

mirroring TH cell subsets . ILC1s express T-bet, produce IFN-

H . GATA-3hi

IL-33, as TH

.

t for their development.

Chapter 1

13

-lymphoid progenitor (

.

CD8+ T cells due to its developmentally dependence on T-bet and Eomes as well as their ability to secrete IFN- , granzyme and perforins . In contrast to ILC1s, NK cells are localized

+ ILC3s can produce IFN as they adopt an ILC1 phenotype by t . Also, NKp46+ +

ILC3san ILC1-like phenotype .

1

14

61 have been characterized in humansresident human ILC precursors, using humanized mice21. Hopefully, further analysis in

ILC development62,63.

64

imprint their gene expression are established very early during development compared to

. In line with this, human

been shown that ILCs are generated in the bone-marrow or fetal liver and then migrate to

466 + ILC3s migrate to

T cells68

resident and maintained by self-renewal in various microenvironments, but hematogenous . Therefore, it is

. Similarly, human adult CD34+ t and an

21. However, the signals that

Chapter 1

cells. Accordingly, mice lacking t .

. Post-birth, LTi cell counterparts also interact with mesenchymal stromal cells to form discrete ILC3 clusters termed cryptopatches . PP are

81 82,83. In more detail, the primary events leading

1 2+ LTi cells and LT + mesenchymal cells, termed

Tumor necrosis factor-a (TNFPP development . Also, PP are absent in mice with simultaneous defects in both CXCL13-

. Cryptopatches predominantly contain adult LTi cells and DCs organized around stromal

cells drives the expression of CCL20 and cells in CPsincluding CCL20 , which recruit B cells , which further grow into mature B cell follicles with the help of TNF produced by DCs and macrophages in response to bacterial compounds

. Mature ILFs generate IgA-producing B cells that target the microbiota, controlling the bacterial community during homeostasis

1 2 by LTi cells . These organized structures provide

1

16

response to damage.

. ILC3s produce 44,100,101. ILC3s are subdivided by the mutually exclusive expression of

+

SILTs . NKp46+

+

as well as HLA+ ILC3s in humans, and these cells are able to regulate CD4+ T cell responses 102.

103

+ ILC3s, through 104

circadian rhythms in the epithelium to control fat storage and other host metabolic

primarily IL-23 and IL-1

. This,

110,111

Chapter 1

Enterococous faecalis and Salmonella spp and 112–114.

. ILC3-derived IL-22 contributes to immunity in a diverse range of clinically-relevant

116, Rotavirus , 118, as well . Extensive research has demonstrated that ILC3s prevent morbidity

in the early phase of cell responses

123,124. Salmonella, for example, has evolved to

.+ and

.

and GM-CSF, in an

manner .

68. MHCII+

+

68,102,130. MHCII+

102,131.

and ILC3s can promote immune responses132

ILC3s .

+ ILC3s and have elevated 133.

responses or aberrant B cell responses, even long before disease onset.

1

18

induces IL-22 expression on ILC3s in the skin. Both 134. It

NKp44 in the skin and blood . ,

138, systemic sclerosis , experimental autoimmune 140

141

ILC3s have also been implicated in the development of cancer, such as in the gut where a 142.

Microbiota-induced IL-1GM-CSF143. GM-CSF maintains CD103+

144. In turn, GM-CSF modulates mononuclear phagocyte 143

128.

by ILC3s , likely through myeloid cells.

Similarly, a new emerging area is the study of ILC3-enteric nervous axis. It has been

of adult ILC3s.

cell-mediated killing in the context of GvHD. And in this thesis, we show that ILC3s contribute

Chapter 1

Lieberkühn

secretory cells .

absorb nutrients and water. Goblet cells secrete mucus-associated molecules that generate a mucus layer to keep microorganisms separated from the epithelium

metabolism

160. Paneth cells are also a source of + ISCs in in vitro cultures, thus being

considered a pivotal component of the ISC niche161

host-defense.

characterized by the expression of the Wnt pathway target gene leucine-rich repeat-+ ISCs

1

20

162

to CBCs, Bmi1+ ISCs are indispensable for epithelial maintenance at steady-state163. They 164 and label-retaining cells or

, mTert , Lrig . However, these genes are broadly expressed by numerous cells in

they have also been termed reserve ISCs

+ cells and Prox1+ cells, as well as enteroendocrine lineage cells possess . In

parallel, another study has reported that Bmi1-GFP+ cells and other secretory cells, including + + + stem

.

.

Gradients of Wnt ligands exist with high expression in crypts and lower expression towards the villi

-catenin signaling pathway. By knocking-out secreted Wnt antagonists, such as , , and the Wnt target gene 180–183

Chapter 1

21

184 Fzd7in vivo

ligands186. Mesenchymal cells, that underlie the epithelium of the crypts, have also been reported to enhance Wnt signaling pathway in ISCs. Even though there is also redundancy when compared to Paneth cells, it is thought that non-epithelial Wnt signals could provide a secondary physiological source of Wnt .

growth factor--catenin suppression. Consequently, BMP levels are higher in

188. At the base of the crypt, mesenchymal-derived . Noggin-1 and Noggin-2, as well as

chordin-like 1 are also highly expressed in the submucosa region by the mesenchyme to suppress BMP signaling .

of epithelial progenitors towards the secretory cell lineage .

and

.

EGF signaling regulates cellular processes that include cell-cycle progression, survival and

demonstrated in the underlying mesenchymal cells and in Paneth cells161. EGF is currently

.

1

22

.

As discussed in the previous paragraphs, there are intrinsic epithelial mechanisms that

cells sense the external forces and respond to the environment200–202

neurons, intraepithelial lymphocytes, smooth muscle cells of the muscularis mucosa, lymph and vascular endothelial cells and a variety of mesenchymal cells, such as pericryptal

203,204

.

.

Chapter 1

23

donor marrow did not have any impact on GvHD-associated organ pathology.

- + + t+

host-derived ILCs appeared to be targeted by the GvHD as their numbers subsided during . Combined

GvHD-induced cell death.

strengthened by the fact that a similar subset of radioresistant NKp46- + + t+ 210.

210. Conversely, absolute levels of IL-22 in the thymus were increased 2 to 3-fold in experimental models of sublethal-TBI without HSCT, lethal TBI and syngeneic HSCT, and T cell-depleted

intrathymic IL-22 was inversely correlated with the thymic cellularity210.

TBI led to an enhancement of thymopoiesis. Thus, the thymic ILC3s are not responsible for

1

24

210

The mechanisms that regulated ILC-derived IL-22 also showed overlap with those found

in IL-23 producing CD103+

The skin also forms a highly organized physical and immunological barrier against the external environment and the microbiota. It is well established that a correct recruitment of

assessed. In an elegant study, ILC3s were shown to favor wound healing by enhancing the

During wound repair, epidermal Notch1 signaling is indispensable for wound closure ,

mediated the recruitment of ILC3s into wounded areas of the skin. ILC3 were required to 211.

and endothelial cells in lymph node anlagen.

Chapter 1

- t+

t+

1

26

AIMS AND OUTLINE OF THE THESIS

The

through which this is achieved.Chapter 1

In

stem cell maintenance post-damage.In chapter 3

+

unknown. Chapter 4

which resembled responses occurring during epithelial damage. This chapter proposes a model that supports a direct link between ILC3s in cryptopatches and the maintenance of

In chapter 5

Finally, a general discussion of the thesis is presented in chapter 6

Chapter 1

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Nature 495,

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36

Dickkopf-1.

c-Myc.

183. van Es, J. H. Dll1+ secretory progenitor cells revert to stem cells upon crypt damage. 14,

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Gastroenterology

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morphogenesis.

Gastroenterology


Nature 519,

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Chapter 1

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206. Lindemans, C. A. Nature

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210. Dudakov, J. A. 336,

7,

1

2

TYPE 3 INNATE LYMPHOID CELLS MAINTAIN INTESTINAL EPITHELIAL STEM CELLS

AFTER TISSUE DAMAGE

Patricia Aparicio-Domingo , Julien J. Karrich1, Ferry Cornelissen1, Natalie Papazian1, Dicky Lindenbergh-Kortleve2, James A. Butler3,

Louis Boon4, Mark C. Coles3, Janneke N. Samsom2 and Tom Cupedo1

1

2

3Centre for Immunology 4Bioceros,

These authors contributed equally

40

ABSTRACT

understood.

severely impaired in the absence of ILC3s or the ILC3 signature cytokine IL-22. These data

cells..

Chapter 2

41

INTRODUCTION

1. Damage sustained by

2 3 and a 4.

epithelium

and in mice, upon DSS-.

12–14

al epithelial cells ex vivo16.

defense against enteric pathogens and containment of microbiota

. Most Nkp46+

21 + ILC3s are located in close 22.

mediated killing23.

42

MATERIALS AND METHODS

Mice t , Ncr1

whenever possible. Thy1+

g every other day, for 2 weeks.

Methotrexate

the last MTX dose.

6 t mice.

exposed to MTX.

Chapter 2

43

and centrifuge at 600 rpm for 3 min to separate the crypts from single cells. Crypts were

+ cells was performed blinded by at least two independent analysts. Pathology score was obtained as previously described (de

and

hours. Protein content of supernatants was determined by enzyme-linked immunosorbent

44

+Lin-NK1.1- + + NKp46

by Flow Cytometry and NKp46+ +

+

+ +

2

Gene Forward Primer Reverse Primer

Gapdh TCAACGGCACAGTCAAG GCTCCACCCTTCAAGTG

GGAGCCCCTTTGTAGACT CGGGAAACTTGCTGTG

CTCCCC CAGTCAGACAG CAATCGCCTTGATCTCTC

GACCCGCCTGAAGATATT ATCCGCATAGGTGGT AAC T

GGGGGCTTCCAGAACT GGGCCATAGAACTGATGAG

CCCCCTGAAACTGGTAGTA GTGGCAGTCTTCAGTTGG

CTTGGCGCAAAAGTGA TTGCTGGATGAGAACAGAA

CAAAAGGATGGTGACATGA GGGTTGTTGACCTCAAACT

Rorc GTGGGGACAAGTCATCTG CGGCCAAACTTGACAG

ACGTCGGGTTGAGAAGA GGATGTGGAGGCTAGATTC

Bax AAGGCCCTGTGCACTAA GAGGCGGTGAGGACTC

CGTGGCCTTTTTCTCC GGCTGCTGCATTGTTC

CGTGGACGCTAGAGAAAG AGCCAGACACTCCATGAC

± SEM.

Chapter 2

RESULTS AND DISCUSSION

cells in S-phase24

. To visualize damage responses by epithelial cells we assessed the number of cycling

+ crypt cells were reduced at day 1 but returned to baseline levels by day 4 . Conversely, transcripts for the

28

in mucosal wound healing8

peaking at day 1 of Stat3 had returned to baseline . These data show that MTX-induced small

. To

are indeed epithelial cell intrinsic or whether immune cells are involved, we administered MTX to mice pre-treated with Thy1-

mice treated with

at day 4

+ mice strongly reduced the recovery of cycling cells at day 4

Stat3 target gene and

mice

46

In conclusion, these experiments establish that

of Thy1+ cells.

+

Chapter 2

+

Gapdh

48

+ cells led us to hypothesize that Thy1+

+ and NKp46+

. Compared + , Rorc, , and , but

and . NKp46+

, Rorc, , , and . These data show that + +

damage by neutralizing IFN mice treated with either neutralizing IFN

. In line with

protein and transcript level . We next exposed Ncr1

pathology. However, the overall pathology in Ncr1

in vivo mice in the presence or absence of Thy1+ cells.

clearly showed that the presence of Thy1+

for , , , and , as well as protein for both IL-22 and GM-CSF

Chapter 2

+NKp46- -NKp46+

mice treated with neutralizing IFN

Gapdh -NKp46+

or Ncr1 Gapdh from

+

tt mice showed impaired recovery of the cycling crypt

t .

Consequently, Stat3 signaling was reduced as evidenced by lower levels of transcripts

.t mice revealed

tt mice at day 4

.

t mice compared to control mice . The extent

t . Survival of epithelial cells

30 t Bax

t Bax-

absence of ILC3s.t+

Chapter 2

Absence of ROR t mice at + t

t Gapdh from t mice t mice at indicated

tt t mice at

day 1 and day 4. Bax and tMTX.

31. Since t

+ ISCs in response to MTX. In order to determine whether +

the percentage of GFP+

+

4 and numbers normalized by day 6 t-expressing

t+ t+

tpathology compared to WT chimeric mice as characterized by crypt

, resembling the pathology t+ lymphocytes on ISCs

+ cells were comparable between both groups

+ ISCs within the EpCAM-1+

+

+

t+ ILC3s as

epithelial cells23

. These data indicate that IL-22 is one of the factors involved in stem

+ t+

Chapter 2

therapies.

+ stem cells within EpCAM1+

t t chimeras at Frequency of GFP+ cells from

WT and t WT and t chimeras at + +

GFP+ cells from WT and tFrequency of GFP+ cells at day 4

(A: 2 independent experiments, N=2 per

of the manuscript. Ncr1

-

Chapter 2

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14,

17,

17,

15,

5, 1–8

15,

10. Anderson, C. A. 43,

19,

12. Gunther, C., Neumann, H., Neurath, M. F. & Becker, C. Apoptosis, necrosis and necroptosis: cell death Gut

Cells. 11,

Nature

Nature 459,

crypt stem cell survival.

Nature 517,

20. Sawa, S., Cherrier, M., Lochner, M., Satoh-Takayama, N., Fehling, H. J., Langa, F., Di Santo, J. P. & Eberl, G.

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23. Hanash, A. M. 37,

194,

28. Miyoshi, H.

Nature 494,

and disease. 17,

Chapter 2

3

GROUP 3 INNATE LYMPHOID CELLS DIRECT

INTESTINAL CRYPTS AFTER TISSUE DAMAGE

Mónica Romera-Hernández1 1 1, 1 1 1,

and Tom Cupedo1

1

2

ABSTRACT

hi

hi ISCs fail to downregulate Wnt and Notch

immune system.

60 Chapter 3

61

INTRODUCTION

1

2,3. + ISC frequencies during in vivo

4. + .

6–8

12,13

.

ILC3s. We show that IL-22 signaling is dispensable for crypt recovery but that in the absence +

3

62


Mice t-GFP, IL-22

mice every 2 days,

i.p to WT mice at day-1 and day 0 for MTX d1 analysis and at day-1, day 0 and day 2 for MTX d4 analysis.

18.

centrifuge at 200g for 2 min to separate the crypts from single cells. Crypts were incubated

Chapter 3

63

Cells were

each gene. Principle component analysis was performed on the fragment counts using the

20.

3

64

+

least two independent analysts. Pathology was scored as previously described21

Chapter 3

+ ILC3s were sorted as indicated.

2

the expression of Gapdhmethod as 2

Primer sequences

Gene Forward Primer Reverse primer

GCCTGATGCTCTTATCTC AAGGCATAGCAGTAGGAG

CCACAAAACAGATGAAGAGAC TCTTTTGCAGCCTCTTATTC

CCATCTTCACGTAGCAGC CAAGATGTCCTGAGGGC


. Samples were analyzed using unpaired Mann-Whitney test. p values <

3

66

RESULTS

t+ ILC3s but are not con-

tt

whereas in IL-22controls

t mice but not of IL-22.

mice during MTX treatment. In these two models,

and STAT3 signaling for stem cell maintenance + . These data show that

t+

t t , IL-22 and IL-22 mice four days

t t , IL-22 and IL-22 mice. t t , IL-22 and IL-22

+

GFP+ mice treated with

+

+

Chapter 3

3

68

, and Gapdh

mice per group.

Chapter 3

hi

MTX d1, including , , Areg, and

treatment, Wnt target genes, such as , , , , , and were

for . This suggests that Yap1 suppresses Wnt signaling, in line with previous reports22. Stem cell

target gene hi

. In line with , 4 . Expression of the receptor Notch ligands and

Besides preserving stemness, Notch also determines cell fate decisions during

+ , and Kitlat MTX d1, and and Kitl further decreased at MTX d4 . Mex3a labels a

+

insults. In our analysis, hi ISCs . Finally, the expression of other

3

which ( + ISCs

and were hi

as , Sct, , , , and . Paneth cell genes, such as , , , , , , Spdef, , Kit and

and Goblet cell genes , , , , and were all upregulated

and returned to steady-state frequencies at MTX day 4, whereas Paneth cell frequencies Alpi

hi

of Bax, , and hi

+

hi

to MTX-induced damage, concomitant to a repression of the Wnt signaling pathway. The

are upregulated, coinciding with an increased frequency of enteroendocrine cells in small

Chapter 3

hi-

hi cells at MTX d1 vs SS and MTX d4 vs SS (DESeq2 FPKM values at SS, MTX d1 and MTX d4 of a selected group

Wnt target genes.

hi SSC-Alo-expressing cells correspond to enteroendocrine cells and CD24hi SSC-Ahi

2fold change (DESeq2

3

Chapter 3

hi

FPKM values at SS, MTX d1 and MTX d4 of a selected group Notch signaling, apoptosis and

stem cells, Paneth cells and Goblet cells. 2fold change (DESeq2 analysis of count

MTX of epithelial stem cells and that Yap1 is important during epithelial remodeling. To test

in vivo we blocked Yap1-

of Yap1 signaling lead to a general increase in pathology . This increased

crypts3

Chapter 3

dependent

thi

hi ISCs display a remarkable divergent gene expression signature in the presence or t+

hi t mice t

. Moreover, comparing the ISC transcriptome at hi ISCs showed 1421 hi t .

hi

t hi

t hi ISCs . Focusing on Yap1 signaling and

, , Areg, and were increased in controls .

hi

t mice t mice .

t t The percentage of crypts t .

hi

t+ cells. , Kitl and were not

t mice t ISCs . Finally,

genes associated with the secretory lineage, including enteroendocrine, Paneth and Goblet

3

t ISCs compared to controls and

hi t, we analyzed the frequencies of

t mice, enteroendocrine cell frequencies were not increased in response to damage .

hi

t-/-

MTX d4

Up Down Down

77 100 22

177 37

hi t t mice 2Fold change values, meaning higher gene expression levels in

2

Control t Control tDown Down Down Down

284 1 2

3

hi ISCs from t t 2Fold change values, meaning increased

2

Chapter 3

hi

hi t thi-expressing

t ISCs when comparing MTX d1 or MTX d4 versus SS. Log2 fold change values for transcripts that correspond to Yap1 and hi-expressing cells comparing MTX

tt

with Yap1+ t Log2 fold change values for transcripts that hi-expressing cells

t Percentages of enteroendocrine cells in duodenal crypt-derived t mice. Enteroendocrine cells were gated as

hi SSC-Alo. Log2 fold change (DESeq2 analysis

3

Log2 fold change values corresponding to the expression hi-expressing cells at MTX d1

tt 2 fold change values were obtained by DESeq2 analysis

group.

. Then, we performed hi

in vivo

treated mice

Chapter 3

+ mice treated , and Gapdh expression

hi- Wnt, as

enteroendocrine cells. Percentages of enteroendocrine

hi SSC-Alo

2

3

80

mice and IL-22FPKM

hi

stem cells, Paneth cells and Goblet cells. FPKM 2

Chapter 3

81

of STAT3. We set out to determine whether Src family kinase signaling is needed for Yap1

in vivo lead to increased total and crypt pathology . Moreover, we interrogated the role of

+

hi

il6ra +

+ ILC3s transcribed , , and

. Similarly, the percentage of . Based on

involvement of L-11 signaling. To test this, we made use of a small molecule inhibitor of

, although crypt pathology and

3

82

hi stem cells at MTX d1. FPKM +

YAP1 immunostaining and percentage of crypts with Yap1+

mice treated with

Chapter 3

83

with MTX d4 of WT mice treated with control or LMT-28 compound.

+ cells in duodenal crypts from WT mice treated with

3

84

DISCUSSION

toxicity associated with cancer treatments.

immune cells, mesenchymal cells and the enteric nervous system, which together form the ISC niche.

+

cells23,24

+ ISC expansion .In this study, we interrogated the in vivo

+

Chapter 3

hi-expressing ISCs in the absence of ILC3s and in vivo

28

22.

30

22. Later during

and this is controlled by increasing Wnt signalling31,32.

+

+ ISCs by Yap1 depends on presence of ILC3s.

mechanism.

30. In agreement with this, our results derived from in vivo

3

86

Based on our data we propose two possible regulatory pathways by which Yap1 mediates + ISCs transcribed Gp130 and il11ra1. Conversely,

in vivofor a possible role of IL-11 in inducing Yap1, which was strengthen with the results observed

+ ISCs is controlled by innate

Chapter 3

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Nature

17,

30. Taniguchi, K. Nature 519,

31. Ashton, G. H. 19,

Nature

Chapter 3

4

INTESTINAL EPITHELIAL STEM CELL HOMEOSTASIS IS ALTERED IN THE ABSENCE OF GROUP 3 INNATE

LYMPHOID CELLS

Mónica Romera-Hernández1 1 1

1 1 and Tom Cupedo1

1

2

ABSTRACT

+

induced damage, independently of IL-22. However, whether presence of ILC3s or IL-22

t but not in in IL-22

was regulated by ILC3s in an IL-22-independent fashion. Since has

+ cells with an enteroendocrine cell phenotype in the absence of ILC3s. When mice were exposed to

+

+ t+ ISC resembling

enteroendocrine cells.

Chapter 4

INTRODUCTION

and consequently shape microbial diversity1

2.

3,4.

+

. ILC3s are an important source of IL-226, which acts mainly on epithelial cells and mesenchymal stromal

of epithelial cells+

10

11. Finally, IL-22 also can act on + 12.

3,13. They are characterized

+ ISCs vulnerable to genotoxic damage14

4

+

such as Bmi1, mTert, Lrig1 or Hopx16–20 + CBCs are completely dispensable

and maintenance of normal crypt-villus architecture 21,22. However, because Hopx and Bmi1

stem cell type remains unknown16–18,21,23

secretory progenitor features.

24. Similarly, recent data suggest that subsets of secretory enteroendocrine +

.

in vivo.

crypt loss. We demonstrate that ILC3s instruct crypt epithelial cells via IL-22 dependent and

hi lo

lo

Chapter 4


Mice t-GFP, IL-22 t-GFP

were used whenever possible.

Methotrexate

mice every 2 days,

centrifuge at 200g for 2 min to separate the crypts from single cells. Crypts were incubated

and counted by using a microscope.

4

genes were performed with DESeq2


30.

performed blinded by at least two independent analysts. Pathology was scored as previously described31

Chapter 4

2


Gene Reverse Primer

CCATCTTCACGTAGCAGC CAAGATGTCCTGAGGGC

GGAAGCCAGTGTCATATCA CCTTGGGGTTCATCTCA

GATGGCTACCGTGGTGT CACCCATGCTCGAATG

GGGGGTGTGAGAATGTCT AGGGCCTTCAGGTCTTC

GCGAAGGGCAAGAATAA TCGGGTCTGTGCTGAG

GCACAACGCACTTTCTTT GGTGGCCCTCAGATGT

TGGACGCTTTGCACTT AGTGGGGGGAAAACTCT

GGAGGACGATGTTCAGATAA CGGCACAGGTAAGAGTTG


4

RESULTS

ROR

degree of bleeding. It also comprised several parameters related to the appearance of the stem cell-containing crypt compartment including degree of crypt loss, crypt abscesses and

stress related transcripts and

controls mice were similar , showing that ILC3 absence does not alter steady-state crypt output. Finally, we examined the in vitro ex vivo isolated

the average number of mature organoids that grew per well . Together these

Chapter 4

t t mice. t t

mice. and Gapdh in total crypts t versus t controls. + t t mice.

t t

4

100 Chapter 4

12,32,33. To

. In line with this, crypt output

mice. General or crypt H&E histological scores in IL-22 mice. +

22 mice.

+

enterocytes are derived from crypt progenitors, we tested whether the presence or absence

+ ISCs express intermediate levels of the cell adhesion molecule CD24 . Hence, we used intermediate CD24 levels (CD24med + ISCs in wild-type B6

t and IL-22hi SSC-Alo hi SSC-Ahi

these three main cellular subsets in absence of ILC3s or IL-22 . Next, we tested + cells at steady-state was controlled by IL-22. To assess

+ ISCs and of the other cell subsets present in

. Even though the frequency of PC was not altered by the absence

t or IL-22 mice. As expected, t and IL-22 PC when compared to WT controls . This is in line with

epithelial cells. In contrast, transcript levels of a marker of both PC and EE cells34 as t mice but not in IL-22 PC

Next, we set out to determine changes in ISC markers and genes important for ISC

med

and the Notch-target gene The Notch-target gene transcribed in CD24med

Notch signaling within crypt epithelial cells enriched for ISCs.

101

4

102

t-/- -/-

reporter mice. t and IL-22 mice. Frequency of CD24med-expressing

t and IL-22 hi-expressing cells, enteroendocrine cells and Paneth cells

within EpCAM+

and Gapdh t and IL-22 mice. and Gapdh med t and IL-22 mice.

Chapter 4

103

+ hi SSC-A

+

t + hi

int cells within total EpCAM-1+

. In line with published reports,

expressing cells t + hi t t mice

t t mice. Genes involved in Notch signaling, such as , and +

t t mice . transcript levels seemed slightly +

+ ISCs expressed similar levels of the regulator of cyclin-dependent kinase, Together these analyses revealed

+

PC, + t t mice

lineage cells (CD24hi SSC-Alo hi int

cytometry t duodenal crypts contained increased frequencies of CD24hi SSC-Alo cells that co-expressed GFP This was the

or intermediate levels . + ISC pool in the absence of

with features of both enteroendocrine cells (CD24hi SSC-Alo +

4

104

t+

t tt + hi int

hi SSC-Alo hi int cells + and hi t t

t Gapdh + cells from t t

Chapter 4

hi

t mice, + t

int

hi cells. In order to gather evidence for either of these hi t t duodenal crypts by

hi ISCs and in line with this high degree of similarity, PCA revealed a poor hi hi t ISCs

hi t crypts when compared to control cells. First, we established the expression of genes involved in controlling stemness, to determine

cells. GFPhi , and

of ILC3s. This indicates that the GFPhi tcell genes. Subsequently we focused on genes associated with mature EE cells, in support

hi cells were enriched for EE cell-associated genes, such as , , , , , Scin,

, and . The increase in EE genes was not due to altered EE cell

enteroendocrine cells in villi or crypts in the absence of ILC3s GPFhi ISCs in the absence of ILC3s co-express secretory genes and stem cell genes, which

int EE precursors.

4

106

hi hi cells at steady-state t t mice. FPKM values of a selected group of genes

associated with stem cells and t t hi cells. +

t t

Chapter 4

+

+ ISCs has been reported in

t thi int ISC

+ CD24hi SSC-Alo

steady-state frequencies of GFP+ EE-lineage cells were increased , but in contrast to control mice, the enrichment of EE-lineage GFP+

completely abrogated discussed in chapter 3. In control mice, GFP+ cells are enriched for EE-lineage genes at day 1

+ . t mice is responsible for this phenotype,

mice. In response to damage and compared to steady-+ cells with the EE-like phenotype was similar

. In sum, these data

+ Frequency of CD24hi SSC-Alo cells hi and int t t mice at SS and

MTX d1. Log2fold change values for transcripts that correspond to genes required during enteroendocrince hi t controls

2 Frequency of CD24hi SSC-Alo cells within hi and int mice treated

4

108

DISCUSSION

stem cells. ILC3s contribute to epithelial renewal in response to damage12,32,33. The cellular

expressing crypt epithelial stem by ILC3s. The reduced levels of

36

of CD24hi SSC-Alo hi int

GFPhi tcells (

, and but similar levels of , , and , among others hi ISCs and mature epithelial cell types,

t under

hi ISC pool to maintain normal crypt output, raising the possibility that survival or hi

possibility, double-immunostaining for secretory and stem cell markers, such as ChgA and

hi ex vivo

for maintenance of crypt homeostasis. IL-22 Paneth cells had no detectable transcript levels of

t and IL-22 mice are explained by

t

Chapter 4

and + + cells in the

in vivo.hi

enteroendocrine cells was increased. These cells acquired secretory cell transcripts and maintained the expression of some stem cell genes. All together this suggests that subsets

int hi

This study contributes to current views on how innate lymphoid cells regulate crypt

+

4

110

REFERENCES

14,

36,

3. Barker, N. Nature 449,

4. Sato, T.

Seminars in

14,

10. Sato, T. Nature 469,

11. Turner, J. E., Stockinger, B. & Helmby, H. IL-22 Mediates Goblet Cell Hyperplasia and Worm Expulsion in 9,


markers. 31,

14. Tao, S. DNA damage. 34,

14,

tumor suppressor. 149,

Science 334,


Chapter 4

111

Nature

14,

Lineage Daughters.

Am J

157,

30. Subramanian, A.

31. de Koning, B. A. E.

32. Hanash, A. M. 37,


34. Sasaki, N.

Nature

36. Jensen, J. Control of endodermal endocrine development by Hes-1.

4

5

DENDRITIC CELLS ASSOCIATE WITH GROUP 3 INNATE LYMPHOID CELLS TO CONTROL INTESTINAL REGENERATION

Mónica Romera-Hernández1 1

1 1, Louis Boon 3 and Tom Cupedo1

1

2

3

114

ABSTRACT

+

CD11c+

that SILTs act as innate control centers where ILC3 and DC interact and translate environmental

+

+ cells contribute to epithelial responses

remain elusive. Next,

+ MHCII+ CD11b- CD103- Plet1+ cells, expressing high levels of and

with ILC3s and able to regulate ILC3. It suggests that SILT-DCs can sense epithelial damage

INTRODUCTION

The mucosal immune system needs to protect the single layer of epithelial cells which is

1,2. DCs induce

the murine small bowel3,4 +CD11b+

. Single CD103+CD11b- cells (CD24+ + CD8 + +

Th1 responses . Another subset of DCs, the CD103-CD11b+ cells are able of inducing both 4 -CD103- cells is a largely

t-

and

by ILC3s is regulated by LT + DCs11

of ILC3 subsets. For instance, DCs expressing CXCL16 retain NKp46+ ILC3 in the LP due to 12,13

.

structures are developmentally dependent on ILC3s and on LT -LT 18

LT and LT mice.

5

116

+ ILC3, DCs and non-

+ ILC3 in SILTs in


Mice t-GFP and hCD2-GFPmice were bred in the animal facility

mice were bred at Icahn School of Medicine at Mount Sinai Hess Center for Science and Medicine

mice. When DT was combined with MTX, DT

as well as in mesenteric lymph nodes.

from hCD2-GFP

in vitro Spleen were excised into small

pressed through the strainer using the plunger end of a syringe. Cells were washed with red

+ cells were

Cells were cultured at 0.3x106

14

5

118

in vitro 6 splenic DCs or BMDC were cultured

Cells were

genes were performed with DESeq2


20.

+

m cell strainers previous

for ILC3s.

5

120

Flow Cytometry and MHCII+ CD11c+ +

were dissected and pierced into small pieces. These were incubated in 2ml of PBS containing

2


Gene Forward Primer Reverse Primer


CTCCCCCAGTCAGACAG CAATCGCCTTGATCTCTC

CGTGGTCCTTGATAACATCT TCACGGCACTGACTGAA

121

RESULTS

CD11c+

induced stem cell damage. We hypothesize that the CD11c+ CP-DCs are involved in this +

all CD11c+

+ cells in the lamina

+ cells in both the LP and MLNs + cells slightly increased,

+

being recruited from bone marrow-derived progenitors. This strategy allowed us to further +

treatment, hypothesizing that CD11c+ cells and ILC3s in cryptopatches are required to

with MTX in mice lacking CD11c+

+ cells per crypt. In the absence of CD11c+

This +

We assessed whether CD11c+ + mice treated with DT or PBS at steady-state. First, we determined +ILC3s within the LP of the SI and found to be unchanged

. Second, we analyzed the level of expression of the signature cytokine IL-22. CD11c+ cell +

that CD11c+

+

5

122

+

Frequency of CD11c+ MHCII+ DCs in lamina propria and in mesenteric lymph

PBS. +

+

mice. Gapdh + ILC3

n=3-6.

123

CD11c+ - -

+ cells that lacked CD103 but expressed Plet1 . We have previously + CD11b+

- Plet1+

t mice. This revealed that CD103- Plet1+ t mice

SILT-DCs. Next, we made use of hCD2-GFP+ reporter mice in which SILTs can be micro dissected using a stereomicroscope due to high expression of hCD2-GFP in ILC3 and B cells. Comparing cell suspensions derived from micro dissected GFP+ SILT regions or GFP- non-SILT lamina propria regions showed that CD103- Plet1+ DCs were only present in GFP+

and absent from non-SILT lamina propria + Plet1- phenotype to isolate CP-DCs and perform phenotypic

macrophages and B cells, and gated as CD11c+ MHCII+

+ DCs are CD11b+CD103+ there is another small subset of CD11b-CD103-Plet1+ DCs

Plet1 . CD103- Plet1+ cells were further examined for the expression of CD11b, CD8

t mice showed decreased frequencies of

+ CD11b- CD103- Plet1+ DCs as SILT-resident cells. 5

124

- - +

t t +CD64-B220-CD11c+MHCII+LiveDead- cells. Frequency of CD103-Plet1+ t t t mice gated as in

. of GFP- and GFP+ + mice. Increase in CD103-Plet1+ DCs in GFP+ +

+CD64-B220 -CD11c+MHCII+LiveDead- in + +CD64-B220-CD11c+MHCII+LiveDead-Plet1+

+CD64-B220-CD11c+MHCII+LiveDead- cells. Histograms show expression of indicated markers -Plet1-

t t +CD64-B220- CD11c+ MHCII+ cells, as DC gate. Frequency of CD103-CD11b- t t

+ CD103- Plet1+ DCs that

upregulated Plet1 transcript levels

DCs increased Plet1 protein expression analyzed Csf2 mice and found that Plet1 transcript levels were almost undetectable in total

. Altogether, these data suggest that

Gapdh in splenic CD11c+ DCs Plet1 expression in bone

Gapdh in ileum from Csf2

5

126

- Plet1+ DC and compared their transcriptome with that of CD11b+ CD103+

regulated in SILT-DCs when compared with CD11b+ CD103+

were found to be down-regulated . Despite the fact that CD11b- CD103- cells

+ DCs are contained within CD11b- CD103-

DCs express lower levels of and + DC development . expression was similar between the two DC subset analyzed. However, SILT-

DCs expressed higher levels of and , which have also been associated with CD11b+

CD103+ and CD11b- CD103+ . Likewise, SILT-DCs expressed and enriched in SILT-DCs, such as and for and +

CD11b- CD8 +

, and . Furthermore, SILT-DCs were enriched for , , , and , whereas they lacked and

. SILT-DCs also expressed high levels of , which indicates that SILT-DCs might be able to migrate to gut-draining lymph nodes. Nonetheless, CD80, CD86 and CD40 were transcribed at similar levels in SILT-DCs and CD11b+ CD103+

. Strikingly, SILT-DCs were enriched for transcripts encoding and ,

DC were and , while and

- + . PCA plot of SI LP CD103-Plet1+

CD11b+CD103+ DCs under steady-state. Heat-map of DESeq2CD103-Plet1+ +CD103+ DCs under steady-state. Blue colors represent the genes with low expression values whereas yellow colors represent the genes with high expression values. FPKM values

DC development, phenotypic DC

helicases, 2

n=3 per group.

5

128

Because CD11c+

MTX-induced stem cell damage

, and , were increased in response to MTX, and the immature DC marker was downregulated

. ,

and , we hypothesized that SILT-DCs might emigrate

CD103- Plet1+ DCs within CD11c+ MHCII+

. Further experiments are in progress to fully demonstrate that SILT-DCs are able to migrate

PCA plot of SI LP CD103-Plet1+ Heat-map of DESeq2 low expression values whereas yellow colors represent the genes with high expression values. Gene expression fold change calculated from DESeq2and

Frequency of DC subsets within CD11c+ MHCII+

5

130

DISCUSSION

+

+

21. To determine whether similar mechanisms

CD103- CD11b-

whether Plet1+

CD103- CD11b- t mice suggests that SILT-DCs are only present when their cryptopatches are formed.

+ CD103- CD11c+

, and , and showed that DC-expressing Plet1 express Csf2-producing ILC3s, likely occurring in cryptopatches.

order to fully elucidate whether SILT-DCs are DC precursors or an intermediate state during

131

22. There is

MLN to induce tolerance - CD11b-

for these diseases.

5

132

REFERENCES

innate immune system.

3. Persson, E.

6,

Cell Homeostasis. 44,

Provide Innate Mucosal Immune Defense.

lymphoid cells. 41,

mucosal draining lymph nodes. 6,

Science

5,

lymphotoxin alpha and the lymphotoxin beta receptor. 173,

157,

133

20. Subramanian, A.

6,

Bacterial Flagellin Enhances Mucosal Innate Immune Defense. 36,

programs. Nature 539,

lymph nodes. 191,

191,

5

6

GENERAL DISCUSSION

SUMMARY

The overall goal of this thesis was to underpin the role of Group 3 Innate Lymphoid cells

+

+

+ ISCs, but

+ ISCs from

+ ISCs likely occurs indirectly via another cell type. We propose that

t

+ ISCs expressing enteroendocrine-like markers, closely resembling a reserve ISC

+

+

+ cells co-localizing with ILC3s in solitary lymphoid follicles and found that they transcribe high levels of

, and

General discussion

138

+

t

Chapter 6Chapter 6

The presence of

+

pathways.

General discussionGeneral discussion

6

140

factors. In the gut mucosa, immune cells reinforce the epithelial barrier and induce tolerance against food or commensal bacteria. Among these immune cells, a recently discovered type of lymphocytes, innate lymphoid cells or ILCs, orchestrates immune responses and maintains

1–3

three types, ILC1s, ILC2s and ILC3s4

IL-22 2-LT 6,8.

.ILC3s also interact with macrophages to establish tolerance towards the commensal

in LysM+

10

towards the commensal microbiota11–14. ILC2s contribute to responses against helminths.

cell death. Epithelial cells sense the danger signals released by the dying cells and produce

18

. However, ILCs can

and ILC3s, which can acquire the ability to make IFN

contribute to gut pathology20,21

from maintenance of barrier, tolerance against commensals and immune responses against

Chapter 6

141

appreciated. ILC3s contribute to skin wound healing22

insult23 24. In this thesis, we

cells, which are crucial for epithelial self-renewal. As a result, epithelial damage is a dose-

lamina propria NKp46+ +

cells , enteric neurons28 and group 3 innate lymphoid cells

+

+

+ CBCs. The cells that + cells, Prox1+

30–34. The actual

General discussion

6

142

+

24.

important results are discussed below. In general, our work provides novel insights into the

vs

. High-dose

In a series of experiments, we demonstrate that ILC3s are necessary to induce a number of

t to demonstrate a role for ILC3s in

wound healingthe peak of damage occurs, which indicates that epithelial responses are induced very

signaling remain to be determined. First, it would be necessary to assess the phenotype of the pSTAT3+

36

enteroendocrine cells (Claudin-4

associated with survival and apoptosis should be assessed. Finally, DNA damage responses

Chapter 6

143

IL-22 induces pSTAT3, as we found that NKp46+

IL-22 neutralized mice vs isotype-treated mice as well as in IL-22

38

+ CBCs mediates stem cell

40.

t

Wnt, Notch, BMP, EGF. During homeostasis, in vitro

General discussion

6

144

t but not IL-22

+ cells leads to a decrease in crypt output, +

t + cell subset that we think interact with ILC3s, therefore it is not a good model to address this. Co-cultures with CD11c+

+ CBCs

+

+ CBCs caused by blocking IL-22 or STAT3 signaling does not translate in reduced

+ CBCs, as supported by previous published reports30,41–43. It might also be possible that de novo +

+

+ CBC maintenance (Chapter 2 and

+

of the stem cell compartment in in vitroIL-22 . However, blocking cytokine signaling or administering recombinant cytokines in vivo or in vitro

+ CBCs.

+ CBCs + CBCs downregulate

Chapter 6

+

damage44 +

reduced, Yap1 is not completely absent in mice lacking ILC3s.

agreement with previous reports that highlighted the importance of Yap1 in crypt epithelial 44–46

epithelial Yap1+ cells in vivo cannot be ruled out.

+ CBCs

important to unequivocally show that Yap1 signaling is regulated in an IL22-independent

. This could

+ CBCs but not

results48.

+ CBCs expressed high levels of transcripts encoding and , and low levels of . Because cryptopatch ILC3s expressed but not , we blocked IL-6 during

General discussion

6

146

IL-6 alone, Yap1+

then be via an indirect mechanism through mesenchymal or epithelial cell-derived IL-11. It t

.

, and

and in response to damage and whether they migrate to lymph nodes to interact with lymph

propose that cryptopatches have emerged in mammals in order to regulate the magnitude

+

Chapter 6

+ .

+ stromal

whether cryptopatches are sites from where epithelial responses are orchestrated.

. Also,

, skin or hair follicles , corneal epithelium60 and lung pulmonary alveoli and bronchi61 + stem cells and can contain ILC3s

6

General discussion

148

necessary. In parallel, in vitro and in vivo experiments could help to elucidate the signals

molecules are produced and signal through membrane-bound receptors. Finally, the candidate molecules, will be also need to be tested both in vitro and in vivo.

Chapter 6

REFERENCES

4. Mortha, A., Chudnovskiy, A., Hashimoto, D., Bogunovic, M., Spencer, S. P., Belkaid, Y. & Merad, M. Microbiota- 343,

Elife 5,

+ T cells. TL - 348. Science

Innate lymphoid cells--a proposal for uniform nomenclature. 13,

8. Balzola, F., Bernstein, C., Ho, G. T. & Lees, C. Innate lymphoid cells drive interleukin-23-dependent innate 11,

Mucosal

14,

Bacterial Flagellin Enhances Mucosal Innate Immune Defense. 36,

13. Macho-Fernandez, E., Koroleva, E. P., Spencer, C. M., Tighe, M., Torrado, E., Cooper, A. M., Fu, Y.-X. &

by epithelial cells.

14. Atarashi, K. 331,

Science 341,

16. Furusawa, Y. cells. Nature

Metabolites produced by commensal bacteria promote peripheral regulatory T-cell Nature

Nature 464,

General discussion

6

Nature 463,

MHCII-mediated dialog between group 2 innate lymphoid cells and CD4+ T cells 41,

21. Nussbaum, J. C. Type 2 innate lymphoid cells control eosinophil homeostasis. Nature

23. Bernink, J. H. 14,

7,

26. Dudakov, J. A. 336,

37,

Development 141,

7,

364,

31. Ibiza, S. Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence. Nature 535,


Lineage Daughters.

Nature 495,

Dll1+ secretory progenitor cells revert to stem cells upon crypt damage. 14,

36. Yan, K. S.

38. Bollrath, J. 15,

Chapter 6


42. Aden, K. 5,

Nature

Genes 7,

13,

Nature 539,

48. Taniguchi, K. Nature 519,

+

13,

Cells. 11,

review.

Mucosal

celiac disease. 356,

PLoS One

39,

17,

64,

General discussion

6

mice.

long-lived, hair follicle stem cells.

adenocarcinoma. 5,

Chapter 6

A

ADDENDUM

ILCs: innate lymphoid cellsILC1s: Group 1 innate lymphoid cellsILC2s: Group 2 innate lymphoid cellsILC3s: Group 3 innate lymphoid cells

GALT: gut-associated lymphoid folliclesLP: lamina propriaBM: bone-marrowPPs: Peyer PatchesCPs: Cryptopatches

LNs: lymph nodesMLNs: mesenteric lymph nodes

CLPs: common lymphoid progenitorsCHILPs: common helper Innate Lymphoid Cell progenitor

SAA: serum amyloid protein

A

Addendum

MNP: mononuclear phagocytes

Plet1: placenta-expressed transcript 1 proteinECM: extracellular matrix

BMT: bone marrow transfer

MTX: methotrexateDSS: dextran sulfate sodium SS: steady-state

YAP1: yes-associated protein 1EGF: epidermal growth factor

Ihh: indian hedgehogGC: Goblet cellPC: Paneth cellEE: Enteroendocrine cellDT: diphtheria toxin

i.p: intraperitoneal

ENGLISH SUMMARY

which cellular and molecular mechanisms are involved in such crosstalk. Second, we also

during these processes.Chapter 1

In , we demonstrated in an experimental model of chemotherapy-induced

+

by ILC3-derived IL-22 in response to epithelial insult in chapter 3. By blocking IL-22 and

damage. This revealed a previously unacknowledged layer of immune cell-mediated control

In chapter 4

t mice at steady-

English summary

A

in chapter 5

Indeed, when we depleted CD11c+

- Plet1+ DCs are present in SILTs and

Finally, chapter 6

to progress this research.

suggest that SILTs are anatomical sites from where innate immune cells orchestrate epithelial

Addendum160

161

Hoofdstuk 1

In beschadiging van de dunne darm aangetoond dat de aanwezigheid van ILC3s in cryptopatches

het epitheel en het repareren van de schade. Dit model van schade aan de dunne darm

zoals bestraling en chemotherapie. In dit model hebben we kunnen aantonen dat ILC3s

+ darm stamcellen na weefselschade.In hoofdstuk 3

is om darm stamcellen te behouden. Echter, de afwezigheid van IL-22 had geen invloed op

van entero-endocriene cellen. Tevens hebben we kunnen aantonen dat de cytokines IL-11

A

162

schade. Deze controle van darm epitheliale cellen door cellen van het immuun systeem was nooit eerder beschreven.

hoofdstuk 4 hebben we bepaald of dit een gevolg was

ook in de afwezigheid van darmschade. Deze veranderingen bestonden onder andere uit crypt-epitheelafvlakking, een verhoogde expressie van stress en ontstekings genen, en een

die, in mindere mate, ook waargenomen werd na toediening van MTX, wat zou kunnen

hoofdstuk 5

+

+

hoofdstuk 6 een algemene discussie over de meest relevante bevindingen

immuun cellen. Het ontrafelen van de mechanismen die zorgen voor ILC3-gemedieerd

AddendumAddendum

163

immuun cellen de homeostase van het epitheel en het herstel van het weefsel controleren. ILC3s zouden daarom in de toekomst wellicht klinisch gemanipuleerd kunnen worden om

te verbeteren.

A

Science and Technology at IES Guillem de Berguedà high school she obtain her cum laude

Fellowship within the Europe’s Erasmus Exchange Programme in order to perform an

PhD candidate in the research group of Dr. Tom Cupedo at the Department of Hematology

th Framework Programme for research and coordinated by Dr. Mark Coles and Prof.dr. Paul Kaye at the Centre for

and the infrastructure of the immune system and collaborated with industrial and academic

Honors:th

Curriculum vitae

A

LIST OF PUBLICATIONS

Romera-Hernandez M,

Mueller CG.

2016.

YH, Mokry M, Romera-Hernandez M, Cupedo T, Dow L, Nieuwenhuis EE, Shroyer NF, Liu C,

damage. Aparicio-Domingo P, Romera-Hernandez M, Karrich JJ, Cornelissen F, Papazian N, Lindenbergh-Kortleve DJ, Butler JA, Boon L, Coles MC, Samsom JN, Cupedo T.

Helder B, Papazian N, Romera-Hernandez M, Tak PP, Cupedo T, Tas SW.

Romera-Hernandez M, Aparicio-Domingo P, Cupedo T.

A

PhD PORTFOLIO

Name PhD student: Erasmus MC Department:

PhD Period:

Promotor:

Supervisor:

Year ECTS

• Imaging course.201320132014

4.244

• Photoshop and Illustrator CS6 Workshop.• The Microscopic Image Analysis course.• Galaxy for NGS.

20132014

0.34

• Work discussions.• Erasmus Hematology Lectures.• PhD lunch with seminar speaker.

• Literature discussions.2012-2016

10

2

2014, 2016

2014

0.61

1.41.41.4

0.3

A

Addendum

Year ECTS

2012-2016

2014, 2016

2014

2

1222

26

1

0.2

6

Acknowledgements

First of all, I would like to thank all the people who have supported me taking this very important step in my career as a PhD student at the Erasmus Medical Center. I have learnt a

discussions. I thank you for teaching me how important is our fundamental research to

To Dr. Tom Cupedo, thanks so much for your great guidance and mentoring as my co-promotor and supervisor. Without your courage and love for science, all this work would

period. I am extremely thankful to you for being always there, so approachable and full of

I want to specially thank Dr. Janneke Samsom for sharing all your knowledge in science,

to convey science into a crystal-clear message.

I cannot be more grateful to any other person in the lab rather than to Natalie. You know it,

Thanks for making me grow as a person next to you. Honestly, I will always have your coco-chocolate sweets as a memory of your special soul.

A

To Ferry, thanks so much for teaching me the importance of being so precise in the lab. I

I am especially thankful to Dicky for showing me everything about immuno-histochemistry.

th

and Linda, it has been a great pleasure to meet you and share experiences in the lab and outside. Thanks for your input during work discussions and your support through all these

Luckily, we will have the chance to discover other parts of the world together, even though

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knowledge with me during work discussions and helping me with machines, reagents and protocols in the lab.

de Bospoort and Jan van Kapel for their support. Also, thanks to Egied for his help in the

Thanks to everyone working at the EDC for taking the best care of our precious mice.

Thanks to my friends and colleagues Emmanuele, Cansu, Almira, Tim, Sophie, Adil, Melissa,

beginning you included me as part of your team and I cannot be happier for that. Thanks for opening your arms to me and show how crazy-fun is life. I will always appreciate you all

years.

for their training during my internship and their guidance and encouragement to start my

Acknowledgements

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career as a PhD.

as an early stage researcher.

shared since my Erasmus in 2011. Jordi, Elena, Lucia, Guillem, Ander, Xabi, Pietro, Joana,

I especially would like to thank my best friend Carla, with who I started this amazing

also to travel around the world with me and always be there when I was sad. I would need

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in Canada.

And last but not least, I am really thankful to the Netherlands that has manage to make me

and specially to your tolerant spirit, which makes possible the coexistence of an amazing

Acknowledgements

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Patching up the crypt - RePub, Erasmus University Repository

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