yersinia-phagocyte interactions during early infection
TRANSCRIPT
Yersinia-phagocyte interactions during early infection
Linda Westermark
Department of Molecular Biology
Umeå Center for Microbial Research (UCMR) Laboratory for Molecular Infection Medicine Sweden (MIMS) Umeå University
Umeå 2013
Copyright © Linda Westermark
ISBN: 978-91-7459-713-4 ISSN: 0346-6612 New series: 1591
Cover picture: Dendritic cell and Y. pseudotuberculosis Printed by: Department of Chemistry Printing Service Umeå, Sweden 2013
To all my loved ones
Follow your heart wherever it may lead you…
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TABLE OF CONTENTS
TABLE OF CONTENTS i
ABSTRACT iii
PUBLICATIONS AND MANUSCRIPTS iv
ABBREVIATIONS v
SAMMANFATTNING PÅ SVENSKA vii
INTRODUCTION 1
1. Yersinia 1
1.1 The Yersinia genus - Species and diseases 1
1.2 Type III secretion system 2
1.2.1 The T3SS apparatus 2
1.2.2 The Yop translocators 3
1.2.3 The Yop effectors 3
1.2.4 The Yop regulators 5
1.2.5 Regulation of T3SS 6
1.3 Adhesins 6
1.3.1 Invasin 7
1.3.2 YadA 7
1.3.3 Ail 7
1.4 Significance of studying Y. pseudotuberculosis 8
2. Phagocytes 9
2.1 Internalization by phagocytosis and micropinocytosis 9
2.2 Monocytes 10
2.2.1 Monocyte subtypes and functions 10
2.2.2 How to study monocytes? 10
2.3 Macrophages 11
2.3.1 Macrophage subtypes and functions 11
2.3.2 Macrophage sensing of pathogens 11
2.3.3 How to study macrophages? 11
2.4 Neutrophils 12
2.4.1 PMN subtypes and functions 13
2.4.2 PMN sensing of pathogens 14
2.4.3 How to study PMNs? 14
2.5 Dendritic cells 14
2.5.1 DC subtypes and functions 15
2.5.2 DC sensing of pathogens 16
2.5.3 How to study DCs? 17
3. Yersinia infection 19
3.1 Infection route and bacterial dissemination 19
3.1.1 Enteric Yersinia infection in humans 19
3.1.2 Enteric Yersinia infection models in mice 19
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3.2 Immune responses during enteric Yersinia infection in mice 22
3.2.1 Yersinia and macrophages 22
3.2.2 Yersinia and neutrophils 23
3.2.3 Yersinia and dendritic cells 24
3.2.4 Yersinia and B and T lymphocytes 24
3.3 Relevance of using mice as a model for human bacterial diseases 25
AIMS 27
RESULTS & DISCUSSION 28
4. PAPER I – Cell type-specific effects of Yersinia pseudotuberculosis virulence effectors 28
4.1 Y. pseudotuberculosis inhibits dendritic cells maturation by the action of YopJ 28
4.2 Y. pseudotuberculosis resists internalization by dendritic cells through the action of YopE 28
4.3 The cell specific effect of YopH depends on the mechanism of internalization 30
5. PAPER II – Immune response to diphtheria toxin-mediated depletion complicates the use of the
CD11c-DTRtg model for studies of bacterial gastrointestinal infections. 33
5.1 DTx-mediated depletion of dendritic cells in the CD11c-DTRtg mouse model induces neutrophilia,
which results in lower Y. pseudotuberculosis infection 33
6. PAPER III – Yersinia pseudotuberculosis efficiently avoids polymorphonuclear neutrophils during
early infection 36
6.1 Y. pseudotuberculosis yopH and yopE mutants, but not the wt strain, interact with PMNs during
early infection 36
6.2 PMN depletion increase the virulence of Y. pseudotuberculosis 37
6.3 Undepleted immature PMNs compensate for the loss of mature PMNs in PMN-depleted mice 39
CONCLUSIONS 41
PAPER I 41
PAPER II 41
PAPER III 41
ACKNOWLEDGEMENTS 42
REFERENCES 44
PAPERS I-III 60
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ABSTRACT
Pathogenic Gram-negative Yersinia species preferentially target and inactivate
phagocytic cells of the innate immune defense by translocation of effector Yersinia
outer proteins (Yops) into the cells via a type III secretion system. This indicates
that inactivation and avoidance of the early innate immune response is an efficient
way for Yersinia species to avoid elimination and to cause diseases ranging from
mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to plague (Y.
pestis). In this project, we aimed to study the interaction between
enteropathogenic Y. pseudotuberculosis and phagocytic cells during early infection.
In situ interaction studies on infected intestinal tissues showed that Y.
pseudotuberculosis mainly interacts with dendritic cells (DCs) in lymphoid tissues of
the intestine during initial infection. After massive recruitment of
polymorphonuclear neutrophils (PMNs) to the infected tissues, wild-type (wt)
bacteria also interacted with this phagocyte. In contrast to the wt, mutants lacking
the anti-phagocytic effectors YopH and YopE are avirulent in mice and unable to
spread systemically. Interestingly, our interaction assay showed that these mutants
not only interacted with DCs, but also with PMNs during the initial stage of
infection. Thus, indicating that Y. pseudotuberculosis can avoid interaction with
PMNs during early infection and that this is Yop-dependent. In a phagocytosis assay
Y. pseudotuberculosis was demonstrated to inhibit internalization by DCs in a YopE-
dependent manner, while both YopH and YopE were shown to be involved in the
blocking of phagocytosis by macrophages and PMNs. Thus, indicating that YopH
has cell type-specific effects. To further investigate the role of DCs during initial
stages of infection, a mouse DC depletion model (CD11c-DTRtg
) was applied.
However, the DTx-mediated depletion of DCs in CD11c-DTRtg
mice induced
neutrophilia and the model could not give a definite answer to whether DCs play
an important role in either restricting or stimulating progression of Y.
pseudotuberculosis infection. To investigate involvement of PMNs during early
infection mice were injected with the depleting antibody α-Ly6G. In absence of
PMNs wt, as well as yopH and yopE mutants became more virulent, which further
supports the importance of these Yops for the ability of Y. pseudotuberculosis to
disseminate from the initial infection sites in the intestine to cause systemic
disease.
In summary, our studies show that inhibiting internalization and maturation of DCs
and avoiding phagocytosis by and interaction with macrophages and PMNs during
the early stages of infection are important virulence strategies for Y.
pseudotuberculosis to be able to colonize tissues, proliferate and disseminate
systemically.
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PUBLICATIONS AND MANUSCRIPTS
This thesis is based on the following publications and manuscript, which are
referred to in the text by their roman numerals (I-III).
I. Cell-type-specific effects of Yersinia pseudotuberculosis virulence
effectors.
Anna Fahlgren*, Linda Westermark*, Karen Akopyan, Maria Fällman.
Cell Microbiol. 2009 Dec;11(12):1750-67. (*Both authors contributed
equally)
II. Immune response to diphtheria toxin-mediated depletion complicates
the use of the CD11c-DTR(tg) model for studies of bacterial gastrointestinal infections. Linda Westermark, Anna Fahlgren, Maria Fällman.
Microb Pathog. 2012 Sep;53(3-4):154-61.
III. Yersinia pseudotuberculosis efficiently avoids polymorphonuclear
neutrophils during early infection Linda Westermark, Anna Fahlgren, Maria Fällman.
Manuscript
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ABBREVIATIONS
Ail Attachment and invasion locus
BM-DC Bone marrow-derived dendritic cell
BM-M Bone marrow-derived macrophage
BM-PMN Bone marrow-derived neutrophil
cDC Conventional dendritic cell
CFU Colony forming unit
DC Dendritic cell
DNA Deoxyribonucleic acid
DTx Diphtheria toxin
DTR Diphtheria toxin receptor
GAP GTPase-activating protein
iDC Inflammatory dendritic cell
IFN Interferon
IL Interleukin
LPS Lipopolysaccharide
IVIS In vivo imaging system
LcrV Low calcium response V
M1 Classically activated macrophage
M2 Alternatively activated macrophage
M cell Microfold cell
MHCII Major histocompatibility complex II
MLN Mesenteric lymph node
MoDC Monocyte-derived dendritic cell
Mreg Regulatory macrophage
NET Neutrophil extracellular trap
NLR NOD-like receptor
PBMC-DC Peripheral blood monocyte-derived dendritic cell
pDC Plasmacytoid dendritic cell
p.i. Post infection
PMN Polymorphonuclear neutrophil
PAMP Pathogen-associated molecular pattern
PPs Peyer’s patches
PRR Pattern recognition receptor
PTPase Protein tyrosine phosphatase
T3SS Type III secretion system
TAM Tumor-associated macrophage
TGF Transforming growth factor
TipDC TNF- and iNOS-producing dendritic cell
TLR Toll-like receptor
TNF Tumor necrosis factor
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YadA Yersinia adhesin A
Yop Yersinia outer protein
YpkA Yersinia protein kinase A
Ysc Yersinia secretion protein
wt wild- type
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SAMMANFATTNING PÅ SVENSKA
Bakterier i Yersinia-genuset infekterar djur och människor och kan orsaka
sjukdomar som sträcker sig från mild gastroenterit (Y. pseudotuberculosis and Y.
enterocolitica) till pest (Y. pestis). De tarmrelaterade Yersinia-infektionerna orsakas
av förtäring av kontaminerad föda och karaktäriseras av diarré, kräkningar,
magsmärtor och feber. Dessa infektioner är vanligtvis självbegränsande hos friska
människor, men kan leda till dödliga, systemiska infektioner hos möss.
När Y. pseudotuberculosis and Y. enterocolitica infekterar via den orala vägen
invaderar de lymphoida vävnader i tarmen via M celler. När bakterierna når
Peyer’s patches och de lymfoida follicklarna i cecum (motsvarar blindtarmen hos
människa) stöter de på immunförsvaret. Försvaret utgörs initialt av makrofager och
dendritiska celler, som har till huvuduppgift att eliminera inkräktare via fagocytos
("cellätande") och starta det specifika försvaret via antigenpresentation. Under
infektionen rekryteras också neutrofiler till infektionsplatsen för att hjälpa
makrofagerna i elimineringen. Väldigt få bakterier kan motstå immunförsvaret i
tarmvävnaden och många infektioner elimineras därför redan här.
Sjukdomsalstrande arter av Yersinia kan dock undvika eliminering genom att stänga
av viktiga immunförsvarsmekanismer (inklusive fagocytos och
inflammationsrespons) och därefter sprida sig vidare till mesenteriska lymfnoder,
mjälte och lever för att orsaka systemisk infektion. Yersinias förmåga att undvika
immunförsvaret är beroende av dess så kallade typ III sekretionssystem (T3SS). Vid
kontakt med en immuncell använder Yersinia sekretionssystemet för att överföra
Yopar (Yersinia outer proteins) till immuncellen. Yoparna stänger av immuncellen
genom att tex blockera fagocytos och mognad, samt orsaka celldöd.
YopH och YopE har identifierats som de två viktigaste Yoparna för att undvika
fagocytos hos makrofager och neutrofiler. YopH är ett effektivt protein tyrosin
fosfatas som stänger av proteiner som är involverade i signalsystemet som är
kopplat till fagocytiska receptorer. YopE har så kallad GAP-aktivitet och kan stänga
av Rho GTPaser som styr många cellulära funktioner, bland annat aktindynamik
som är viktigt för fagocytos. YopJ är ett serin/threonin acetyltransferas som stänger
av olika signalvägar i immuncellerna och påverkar till exempel celldöd och
cytokinproducering i infekterade celler.
I vår första studie visade vi att Y. pseudotuberculosis kan stänga av
mognadsprocessen hos dendritiska celler med YopJ och att bakterien kan undvika
fagocytos. De anti-fagocytiska Yoparna hos Y. pseudotuberculosis visade sig ha
cellspecifika effekter eftersom bakterien använder både YopH och YopE för att
undvika fagocytos hos makrofager och neutrofiler, medan endast YopE har en
effekt mot dendritiska celler. Detta visade sig beror på att dendritiska celler har en
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annorlunda mekanism för att internalisera bakterier, som inte involverar
receptorsignalering.
I den andra studien fortsatte vi undersöka hur Y. pseudotuberculosis interagerar
med dendritiska celler genom att använda en musmodell (CD11c-DTRtg
) i vilken
man kan ta bort dessa celler genom att behandla mössen med difteritoxin. Tyvärr
visade det sig att stora mängder neutrofiler kom till vävnaden där de dendritiska
cellerna tagits bort och att dessa sedan eliminerade bakterierna när de försökte
infektera mössen. Genom att förskjuta infektionen två dagar, tills de flesta
neutrofilerna hade försvunnit kunde vi konstatera att dendritiska celler
förmodligen inte spelar en särskilt viktig roll under de tidiga stadierna av Y.
pseudotuberculosis-infektion. Vi drog också slutsatsen att denna modell måste
användas varsamt vid infektionsstudier och att sådana inte kan initieras förrän
immunresponsen har gått tillbaka efter behandlingen.
I den tredje studien tittade vi på hur Y. pseudotuberculosis interagerar med
immunceller i de lymfoida vävnaderna i tarmen under de första stadierna av oral
infektion. Det visade sig att vildtypsbakterier initialt bara interagerar med
dendritiska celler och först senare med neutrofiler. Mutanter som saknar de anti-
fagocytiska Yoparna, YopH eller YopE interagerar med dendriska celler lika ofta som
vildtypen, men interagerar också med neutrofiler. Detta skulle kunna förklara
varför yopH-och yopE-mutanter inte kan sprida sig systemiskt och orsaka sjukdom.
För att studera detta vidare injicerade vi möss med antikroppar som tar bort
neutrofiler och undersökte hur detta påverkade infektionen. Det visade sig att
vildtypen, men framför allt mutanterna blev bättre på att infektera mössen om
neutrofiler inte fanns. Resultaten visar att Y. pseudotuberculosis interagerar med
fagocytiska celler i tarmen under de första stadierna av oral infection, men att
Yoparna gör bakterien tillräckligt effektiv för att kunna undvika immunförsvaret,
sprida sig systemiskt och orsaka en infektion med dödlig utgång. Om bakterien
däremot saknar någon av de viktiga Yoparna blir den stoppad och kan inte sprida
sig systemiskt. Neutrofiler verkar vara ansvariga för denna blockering eftersom
mutanterna kan sprida sig snabbare i möss som saknar neutrofiler.
Sammanfattningsvis visar våra studier att Y. pseudotuberculosis genom att
förhindra internalisering och mognad av dendritiska celler och genom att undvika
fagocytos och interaktion med makrofager och neutrofiler i ett tidigt stadium under
infektionen, kan kolonisera vävnader, föröka sig och sprida sig vidare till inre organ.
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INTRODUCTION
1. Yersinia
1.1 The Yersinia genus - Species and diseases
The Yersinia genus consists of Gram-negative, rod-shaped bacteria. The genus is
named after the Swiss bacteriologist Alexandre Yersin, who independently of
Shibasaburo Kitasato successfully isolated and described Y. pestis in Hong Kong in
year 1894, during the third plague pandemic [1]. The Yersinia genus includes in
total 15 identified species, represented among environmental, commensal, as well
as pathogenic bacteria. Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica are
the extensively studied and characterized human pathogenic Yersinia species. They
harbor a 70-kb virulence plasmid (pYV) found in all disease causing strains, but not
in the other Yersinia strains [2]. Y. frederiksenii, Y. intermedia, Y. kristensenii, Y.
bercovieri, Y. mollaretii, Y. rohdei, Y. ruckeri, and Y. aldovae are considered to be
non-pathogenic to humans. They are sometimes referred to as Y. enterocolitica-like
bacteria, and have not been studied to the same extent. However, the majority of
these species have been isolated from humans and animals, and it is suggested that
some of them may cause disease with virulence factors different from Y.
enterocolitica [3].
Y. pestis is the causative agent of plague and is responsible for several severe
pandemics and the death of millions of people throughout history. Even though
large plague pandemics do not arise today, small outbreaks of plague still occur in
parts of Asia, Africa and America. Nowadays, Y. pestis is also considered to be a
major treat as a bioweapon due the serious disease it causes and to the occurrence
of antibiotic resistant species. Y. pestis spread via bites from infected Xenopsylla
fleas or aerosols and cause bubonic, septicemia or pneumonic plague depending
on the way of transmission. Unless treated with antibiotics, the mortality rate is
very high [2].
The enteric Yersinia species, Y. pseudotuberculosis and Y. enterocolitica are the
most commonly encountered. They can be found in aquatic environments, soil and
animals, and cause the disease yersiniosis worldwide. Yersiniosis is a
gastrointestinal disease characterized by symptoms like fever, abdominal pain,
vomiting, and diarrhea. Infections are spread through contaminated food and
water, and are usually self-limiting in humans [2]. However, enteric Yersinia
infections have been linked to human inflammatory autoimmune diseases, e.g.
Crohn’s disease and reactive arthritis [4, 5], and Y. pseudotuberculosis infections
have also been connected to Far East scarlet-like fever and Kawasaki disease [6, 7].
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1.2 Type III secretion system
The ability of bacteria to deliver virulence effectors into host cells is essential for
infection, survival and pathogenesis for many Gram-negative bacteria. Bacteria use
eight different so called secretion systems to transport proteins over their cell wall.
The cell wall of Gram-negative bacteria consists of two membranes, the inner and
outer membrane. Many pathogenic Gram-negative bacteria use a type III secretion
system (T3SS) to transfer proteins from their cytoplasm, over both membranes, to
the cytosol of target host cells [8]. In Yersinia the secretion system was first
discovered in 1990 by Cornelis and coworkers [9], but it was not denoted a T3SS
until Reeves and Salmond discovered the homology between proteins in the
Yersinia secretion system and proteins expressed by plant pathogens [10]. The
whole Yersinia T3SS, including proteins of the secretion apparatus, translocation
proteins, secreted proteins and regulatory proteins are encoded on a virulence
plasmid present in all pathogenic species [11].
In Yersinia the proteins secreted by the T3SS are called Yersinia outer membrane
proteins or Yops, and they can be divided into translocators (YopB , YopD, and
LcrV), effectors (YopH, YopE, YopJ/P, YopK, YopM, YopT, and YpkA/YopO), and
regulators (YopK/YopQ and YopE). The name was originally given because the Yops
were discovered before the secretion system, and were then found localized in the
outer membrane of Y. pseudotuberculosis and Y. enterocolitica [12, 13].
1.2.1 The T3SS apparatus
The T3SS apparatus is often structurally referred to as a syringe, injectisome, or a
needle-like nanomachine. The Ysc-T3SS apparatus of Yersinia is no exception. The
apparatus is build up by more than 20 different proteins forming a membrane
embedded basal structure, an external needle protruding from the bacterial
surface, and a tip complex that caps the needle [14, 15]. In Yersinia the basal
structure is build up by a number of Yersinia secretion proteins (Ysc), e.g. the
scaffold proteins YscCDJ and the export apparatus YscRSTUV. The needle is formed
by YscF and the tip complex contains LcrV.
The similarity between the needle complex and a syringe has generated a generally
accepted model that effectors are directly injected from the bacterial cytosol into
the cytoplasm of the targeted host cell. Nevertheless, there are no experimental
evidences showing that the effectors are secreted through the needle structure.
Instead a study visualizing the presence of Yop translocators and effectors on the
bacterial surface before target cell contact challenge this injectisome model. This
study proposes a two-step translocation model in which the T3SS substrates are
first secreted to the bacterial surface and thereafter translocated across the target
cell membrane upon contact [16].
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1.2.2 The Yop translocators
Upon contact with host cells, Yop translocators (YopB and YopD) are assembled
into a translocon between the tip complex (LcrV) of the needle and the host cell
membrane. The translocon will form a pore in the host cell membrane and work as
a gateway for translocation of effector proteins [14].
1.2.2.1 YopB and YopD
YopB and YopD are transmembrane proteins responsible for forming the
translocation pore in the host cell membrane [17]. The two proteins bind each
other and are both required for pore formation to occur [18, 19]. Thereby, YopB
and YopD mediate the translocation of effector Yops into the host cell [20, 21].
Both yopB and yopD mutants are avirulent in mice [22, 23].
1.2.2.2 LcrV
Besides YopB and YopD, LcrV (Low calcium response V or the V antigen) has been
shown to be required for translocation of effector Yops [24-26]. LcrV has been
localized at the bacterial surface and can be found at the tip of the YscF needle [27,
28]. The protein is involved in the release of the pore forming proteins YopB and
YopD from the bacterium and in determining the size of the pore, but is not a
structural part of the Yop translocation pore [29, 30]. LcrV is necessary for the
virulence of Yersinia [31]. It is a protective antigen for infection by all three human
pathogenic Yersinia species and has been extensively studied as a vaccine
candidate against Y. pestis [32, 33].
1.2.3 The Yop effectors
The T3SS apparatus and the Yop translocators provide the means for infection, but
the real action is performed by the proteins secreted by the system – the Yop
effectors. In Yersinia there are six common Yop effectors: YopH, YopE, YopT YopJ/P,
YopM, and YpkA/YopO. After translocation into the host cell cytoplasm, the
effector proteins initiate and maintain infection by interfering with important host
cell functions, e.g. cell signaling, phagocytosis, and inflammatory responses [34].
1.2.3.1 YopH
YopH is a very potent and fast acting protein tyrosine phosphatase (PTPase) [35,
36]. After translocation YopH localizes to peripheral focal adhesion complexes in
the host cell [37, 38] and targets proteins involved in receptor signaling and
adaptor proteins, such as FAK, p130Cas, Fyb (also called ADAP and SLAP-130), and
SKAP-HOM [37-40]. Thereby, YopH inhibits Ca2+
signalling in neutrophils and
disrupts focal adhesion complexes resulting in inhibition of phagocytosis and the
associated oxidative burst in macrophages and neutrophils [37, 38, 41, 42]. YopH
has also been shown to target LAT and SLP-76 in T lymphocytes and suppress the
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activation of B and T lymphocytes [43, 44]. Thus, YopH might have different targets
in different cell types. Certainly, this PTPase performs many important actions
during infection and subsequently, a yopH mutant is avirulent in mice [45, Paper
III]. The role of YopH in the strategies of Yersinia to avoid the immune defense will
be further discussed in section 3.2.
1.2.3.2 YopE
YopE is a GTPase-activating protein (GAP) that acts on GTPases of the Rho family
[46-48], which are important regulators of actin dynamics in eukaryotic cells [49].
In vitro YopE has been reported to stimulate GTP hydrolysis of Cdc42, RhoA and
Rac1 and thereby inhibit these proteins [46, 47]. However, inside cells YopE
preferentially inactivates Rac1 and RhoA, but not Cdc42 [48]. The GAP activity of
YopE is required for the anti-phagocytic capacity and virulence of Yersinia [46]. The
role of YopE as a regulator of Yop secretion and in the strategies of Yersinia to
avoid the immune defense will be further discussed in sections 1.2.4.2 and 3.2,
respectively.
1.2.3.6 YopT
The cysteine protease YopT inactivates Rho family GTPases by removing lipid
modifications [50]. In vitro, YopT can efficiently cleave recombinant RhoA, Rac and
Cdc42, while RhoA is the preferred substrates inside infected cells [51, 52]. YopT
has been reported to contribute to the anti-phagocytic ability of Y. enterocolitica
against phagocytic cells [51, 53, 54]. However, a Y. enterocolitica yopT mutant is as
virulent as the corresponding wt strain in mice [55, 56], showing that YopT is not
essential for causing disease. Further supporting this is the fact that serotype O3
strains of Y. pseudotuberculosis, which do not express YopT because of a deletion
on the virulence plasmid, are still virulent in mouse infection models [57]. A
comparative study of YopT and YopE showed that YopE is more efficient than YopT
in blocking phagocytosis, MAPK and NFκB signaling pathways and interleukin-8 (IL-
8) production [58]. Taken together, these observations suggest that YopT function
might be redundant due to the similar function of YopE.
1.2.3.5 YpkA/YopO
YpkA (YopO in Y. enterocolitica) is a multifunctional protein that can function as a
serine/threonine kinase and has both a Rho-GTPase binding domain and an actin
binding domain. [59-63]. YpkA can disrupt the actin cytoskeleton and thereby
contribute to the anti-phagocytic ability of Yersinia against macrophages [54, 64,
65]. However, our investigations do not show any contribution of YpkA to the anti-
phagocytic capacity of Y. pseudotuberculosis against either macrophages or DCs
(Fällman and Westermark, unpublished data). Also when it comes to the
importance of YpkA for virulence in mice results differ, and ypkA mutants have
5
been reported to be both avirulent [59, 65, 66] and as virulent as wt bacteria [55,
67].
1.2.3.3 YopJ/P
YopJ, or YopP as the protein is named in Y. enterocolitica, is a serine/threonine
acetyltransferase [68, 69]. By inhibiting several signaling pathways YopJ interferes
with apoptosis and cytokine production in infected cells [70-74]. The molecular
targets of YopJ/P have been identified as MAPK kinases (MKKs) and IkB kinase-β
(IKKβ) [73]. YopJ/P has been shown to be important for virulence of
enteropathogenic Yersinia [55, 75, 76], but dispensable for virulence of Y. pestis
[77, 78]. However, others have reported similar results for YopJ in Y.
pseudotuberculosis, i.e. that deletion of YopJ had no effect on virulence [66, 79].
The discrepancy regarding the importance of YopJ/P for virulence might be
explained by the difference in cytotoxic activity reported for YopJ/P proteins from
different Yersinia species and even serogroups. High or increased cytotoxicity of
YopJ/P is suggested to lead to proinflammatory responses and avirulence [76, 80,
81]. The role of YopJ/P in the strategies of Yersinia to avoid the immune defense
will be further discussed in section 3.2.
1.2.3.4 YopM
The functional role of YopM has been somewhat a mystery for a long time. One
reason might be that YopM is the only Yop effector that lacks an enzymatic activity
[57]. YopM is a leucin rich repeat protein that has been reported to have a nuclear
localization in host cells [82-84]. This protein functions as an adaptor protein by
forming a complex with and activating two cytoplasmic kinases, RSK1 and PRK2 in
J774 macrophages and HEK 293 cells [85]. YopM interferes with the immune
response of the host by changing the cytokine response, i.e. induce expression of
the anti-inflammatory IL-10 [86]. Interaction of YopM with RSK1 and PRK2 is
required for the induction of IL-10. Recently, YopM was shown to directly inhibit
caspase-1 in macrophages, which is an important participant in the inflammasome
[87]. This finding might explain why YopM is an important virulence factor and that
a yopM mutant is avirulent in mice [55, 88-90]. The role of YopM in the strategies
of Yersinia to avoid the immune defense will be further discussed in section 3.2.
1.2.4 The Yop regulators
1.2.4.1 YopK/Q
YopK (YopQ in Y. enterocolitica) is secreted and translocated together with the
effector Yops into the targeted host cell. However, this Yop does not have a direct
activity against the host cell itself, but instead a regulatory role for the Yop
secretion and translocation. This function was discovered as a yopK mutant delivers
increased levels of Yop effectors into infected cells [91]. YopK interacts with the
6
pore-forming protein YopD and regulates the ratio of YopB and YopD in the target
cell membrane. Consequently, a yopK mutant forms larger pores, which results in
the over-translocation phenotype [92]. The interaction of YopK with Rack1 in the
host cell is important for full anti-phagocytosis and appears to direct the action of
the effector Yops to the correct locations in the cell [92]. A precise and controlled
Yop delivery seems to be very important for Yersinia virulence, since a yopK mutant
is avirulent in mice despite its over-translocation phenotype [55, 92-95]. The role of
YopK in the strategies of Yersinia to avoid the immune defense will be further
discussed in section 3.2.
1.2.4.2 YopE
In addition to its role in anti-phagocytosis, YopE has been reported to be involved
in the regulation of Yop translocation together with YopK. Similar to a yopK mutant,
a yopE mutant delivers increased levels of Yop effectors into infected cells [48, 96].
Since both yopE and yopK mutants over-translocates Yop effectors, it has been
suggested that they work together for a controlled Yop translocation [96]. The role
of YopE in the strategies of Yersinia to avoid the immune defense will be further
discussed in section 3.2.
1.2.5 Regulation of T3SS
To be successful in virulence bacteria need to express the right set of proteins at
the right time, and at the same time be able to control the use of these proteins. In
Yersinia the T3SS is regulated on many different levels. The expression of the T3SS
and its effector proteins is regulated by temperature, calcium concentration and
host cell contact. The T3SS expression is repressed at 26°C and high calcium
concentration, but expressed at 37°C, low calcium concentrations and upon host
cell contact [77, 97-103]. Secretion and translocation of Yop effectors are regulated
by host cell contact and by two of the effector proteins themselves (YopE and
YopK, also mentioned in section 1.2.4) [21, 103]. In addition, some Yops have
chaperones, which further control the secretion [104].
1.3 Adhesins
During different stages of infection it is required for invading bacteria to be able to
adhere to host cells. Adhesion is mediated by outer membrane proteins called
adhesins. Pathogenic Yersinia species express a number of different adhesins. This
section will present three of these adhesins, expressed by the enteropathogenic
Yersinia species: invasin, yersinia adhesin A (YadA) and attachment and invasion
locus (Ail) [2].
7
1.3.1 Invasin
Invasin is encoded on the chromosome and has its optimal expression at 26°C and
basic conditions or under acidic conditions at 37°C [105]. Invasin is thought to be
present on the bacterial surface during the initial phase of infection, when Yersinia
enters the small intestine [106] and to facilitate the entry into the intestinal tissue
by binding to β1-integrins expressed on the apical surface of microfold cells (M
cells) in the follicle associated epithelium [107-110]. Studies have shown that
invasin promotes the initial steps of host colonization and penetration of the
intestinal epithelium, but have no effect on establishment of a systemic infection
as an invasin mutant can cause fatal infection [111-113]. Invasin can promote Yop
delivery upon contact with host cells [114-116]. However, the interaction between
invasin and β1-integrins is not only beneficial for the bacteria, since it can also
mediate uptake by macrophages [117, 118, Paper I].
1.3.2 YadA
YadA (Yersinia adhesin A) is encoded on the virulence plasmid and exclusively
expressed at 37°C in high levels [119]. Thus, YadA is the major adhesin expressed
after enteric Yersinia has crossed the intestinal epithelial barrier. This protein has
multiple functions. As an adhesin it binds extracellular matrix proteins (collagen,
fibronectin, and laminin) and cells (including macrophages and epithelial cells)
[120] through indirect binding to β1-integrins via a bridging extracellular matrix
molecule [121]. YadA is also the major contributor to Yersinia serum resistance, i.e.
the ability to avoid killing by the complement system [122]. The dense layer of
YadA on the bacterial surface is thought to function as a physical barrier that
prevents the complement systems from opsonization and membrane attack
complex formation. Further, YadA binds two host serum factors responsible for
regulating the activation of the complement system: C4 binding protein and factor
H. C4 binding protein is a negative regulator of the classical pathway [123] and
factor H regulates the alternative pathway by stimulating the cleavage of C3b to its
inactive form iC3b [124, 125]. Thus, YadA enables Yersinia to avoid killing by both
the classical and the alternative complement pathways. A yadA mutant is poor in
colonization and persistence in the PPs of mice [109, 111, 113]. However, this
adhesin does not play an important role for the overall infection since the YadA
deficient YPIII strain of Y. pseudotuberculosis is not attenuated and cause lethal
disease in mice [93, 126].
1.3.3 Ail
Ail (Attachment and invasion locus) is chromosomally encoded and expressed at
37°C under aerobic conditions (Pierson and Falkow 1993). Ail mediates epithelial
cell binding and invasion like invasin, but only to certain cell types, such as HEp-2,
HEC1B, and CHO cells (Miller and Falkow 1988). This might reflect a restricted
8
expression of Ail receptors. Similar to YadA, Ail is involved in serum resistance by
binding C4 binding protein and factor H [123, 127, 128], and thereby inhibits both
the classical and the alternative complement pathways.
1.4 Significance of studying Y. pseudotuberculosis
According to statistics from the Swedish Institute for Communicable Disease
Control approximately 300-800 cases of enteric Yersinia infections per year were
reported in Sweden 2003-2012, corresponding to 3-9 cases per 100,000 habitants
per year. This low prevalence of disease might raise the question why study Y.
pseudotuberculosis? First of all, Y. pseudotuberculosis is an optimal model for
studies of microbial pathogenesis and host responses. It is easy to handle and
cultivate, which is a basic requirement for scientific studies of a bacterium. Its
genome is sequenced and genetic manipulations of this bacterium are greatly
simplified since many of the virulence factors expressed by this pathogen are
encoded on the virulence plasmid. Moreover, scientists interested in studying
bacterial virulence accentuate the close relationship with the more dangerous Y.
pestis, but also that Y. pseudotuberculosis share common virulence traits with
other pathogens e.g. the T3SS. For others, Y. pseudotuberculosis is a good model to
study host-microbe interactions, as it offers a virulence model in mouse for both
acute and persistent infection. Additionally, the effector proteins are directed
against important immune cell functions and mutants can be used to study these.
Taken together, Y. pseudotuberculosis is a good model organism for studying
various aspects of pathogenicity and immune response, including adhesion,
effector protein delivery into host cells, immune evasion, and immune cell
functions.
9
2. Phagocytes
Phagocytes are cells with phagocytic capacity i.e. the ability to internalize particles.
Internalization of larger particles can either occur by phagocytosis or
macropinocytosis. The innate immune system includes a number of phagocytes:,
monocytes, macrophages, neutrophils, and dendritic cells [129].
To sense their surroundings phagocytes express a number of different receptors
collectively called pattern-recognition receptors (PRRs). These receptors recognize
conserved molecules called pathogen-associated molecular patterns (PAMPs) from
invading fungi, viruses, bacteria, and parasites [130].
2.1 Internalization by phagocytosis and micropinocytosis
Phagocytosis, which means cell eating, is a highly conserved and complex
mechanism for particle internalization. Phagocytosis is often mentioned in the
context of elimination of foreign particles and microorganisms, but is also used to
remove dangerous “self” cells and debris generated during development, tissue
turnover and repair [129]. Phagocytosis is a multi-step, receptor-mediated, actin-
dependent process that proceeds in a zipper-like or sinking manner [131].
Internalization through phagocytosis often requires and is initialized by recognition.
The recognition is mediated by direct or indirect binding of the particle or
microorganism to surface receptors on the phagocytes. Direct binding occurs
between surface structures on the phagocytized object and a phagocytic receptor
on the phagocyte, for example interaction between sulfated and mannosylated
sugars and the mannose receptor. In contrast, indirect recognition occurs through
opsonins, for example IgG antibodies or complement fragments, bindning to Fcγ-
receptors and complement receptors, respectively [129]. After recognition by
receptor ligation, signaling from the phagocytic receptor and subsequent local actin
polymerization is activated. This cause internalization and results in formation of a
phagosome in the cytosol of the phagocytic cell [129]. The phagosome then
undergoes maturation through interaction with early and late endosomes and
lysosomes, to finally transform into a phagolysosome. Inside the phagolysosome
ingested particles and microorganisms are degraded through acidification and
enzymatic activities [132].
Macropinocytosis is a clathrin-independent form of endocytosis that mediates
non-selective uptake of solute molecules, nutrients, antigens and even
microorganisms [133]. Like phagocytosis, macropinocytosis is an actin-dependent
process, but instead of proceeding in a zipper-like manner, it is initiated from
surface membrane ruffles. Another difference is that macropinocytosis can give
rise to large endocytic vacuoles called macropinosomes. After formation, the
macropinosomes undergo maturation and fuse with lysosomes. Normally
10
macropinocytosis is a signal-dependent process that occurs in response to growth
factor stimulation, by for example CSF-1 or EGF, but in antigen-presenting cells this
uptake process is constitutively active [133].
2.2 Monocytes
Monocytes develop from bone marrow precursors and circulate in the blood.
During both steady-state and inflammatory conditions monocytes can migrate
from the blood into tissues and differentiate into different types of macrophages
[134] and dendritic cells (DCs) [135, 136]. Monocytes can also directly mediate
antimicrobial activity in infected tissues by release of antimicrobial substances and
transport of antigens to draining lymph nodes, where they either transfer the
antigens to conventional DCs (cDCs) or differentiate into monocyte-derived DCs
[137]. Thus, monocytes are multifunctional cells with roles in homeostasis, immune
defense, and tissue repair.
2.2.1 Monocyte subtypes and functions
In both humans and mice monocytes are divided into three subsets: classical,
intermediate, and non-classical. I humans expression of CD14 and CD16 is used to
distinguish the different subtypes. Classical monocytes are CD14++
CD16-,
intermediate monocytes are CD14++
CD16+, and non-classical monocytes are
CD14+CD16
++. In mice expression of Ly6C and CD43 is used to separate the
subtypes as classical monocytes are Ly6C++
CD43+, intermediate Ly6C
++CD43
++ and
non-classical Ly6C+CD43
++ [138].
As their differential gene expression patterns indicate, monocyte subsets also differ
in their function. Non-classical monocytes show a patrolling behavior in the blood
vessels and are involved in tissue repair, while classical monocytes is the major
subtype of monocytes being recruited during early inflammatory responses [139].
2.2.2 How to study monocytes?
The high numbers of monocytes in blood and bone marrow makes monocytes
possible to isolate. However, the lack of a monocyte-specific marker complicates
identification of monocytes and enforces the use of combined markers and
preferably flow cytometry.
2.2.2.1 Monocyte depletion models in mouse
Since there are no monocyte-specific markers known, there is also no specific
monocyte depletion model. However, since monocytes express Ly6C, they can be
depleted together with neutrophils by injection of α-Gr-1 antibody (further
discussed in section 2.4.3.2).
11
2.3 Macrophages
Macrophages are professional phagocytes located in nearly all tissues of the body.
Macrophage precursors are released from the bone marrow into the blood
circulation as monocytes, which migrate into tissues and differentiate into mature,
tissue-resident macrophages. Macrophages are often strategically located to
perform their important immune surveillance activities against commensals and
possible invaders. These activities include phagocytosis, antigen presentation,
immune suppression during antimicrobial defense, anti-tumor immunity, and
inflammatory responses [140].
2.3.1 Macrophage subtypes and functions
Generally it is believed that macrophages represent a variety of activated
phenotypes rather than separate subpopulations [140]. The large overlap in
expression of surface markers between different subsets of macrophages have led
to the use of quantification of specific gene expression profiles after cytokine or
microbial stimulation, to characterize this cell type. Consequently, macrophages
have been divided into classically activated (M1), alternatively activated (M2),
regulatory (Mreg), and tumor-associated macrophages (TAMs). The classically
activated macrophages mediate defense against bacteria, protozoa and viruses,
and further play a role in the immune response against tumors, while the
alternatively activated macrophages have anti-inflammatory activity and regulate
wound healing. The regulatory macrophages are regulatory because they secrete
large amounts of IL-10 and the tumor-associated macrophages suppress anti-tumor
immunity [140].
2.3.2 Macrophage sensing of pathogens
To be able to fulfill their function as sentinel cells, macrophages express a large
variety of PRRs, including Toll-like receptors (TLRs), C-type lectin receptors,
scavenger receptors, mannose receptors, retinoic acid-inducible gene 1-like
helicase receptors and NOD-like receptors (NLRs) [140]. In macrophages these
receptors are not only involved in the recognition of PAMPs on invading
microorgansims, but also foreign substances and dead or dying cells. In addition,
macrophages express complement and Fc receptors that bind to opsonizing
molecules, i.e. C3b and antibodies bound to the surface of invading
microorganisms and facilitate phagocytosis [140].
2.3.3 How to study macrophages?
Macrophages are present in many different tissues, but as for DCs the numbers are
quite low. In mouse PPs, MLN and spleen the number of macrophages is around 1%
of the total number of leukocytes [111]. Isolation procedures are therefore
inefficient, expensive, and time consuming. Alternative methods, such as use of
12
macrophage cell lines, in vitro differentiation from precursor cells and depletion
models are therefore widely used to study macrophages. Expression of the
glycoprotein F4/80 is commonly used to identify macrophages. Further, differential
expression of F4/80 in combination with CD11b, CD18, CD68 and Fc receptors is
used to distinguish macrophages from DCs [140].
2.3.3.1 Macrophage cell lines
The J774 and Raw264.7 cells are derived from tumors in BALB/c mice and are
macrophage-like cell lines widely used for macrophage studies. Both cell lines are
easy to culture and can be used to study phagocytosis and cytokine production
[141].
2.3.3.2 Macrophage differentiation models in mouse and human
Mouse macrophages can be differentiated from bone marrow progenitors by
stimulation with the growth factor M-CSF [142]. Human blood monocytes can be
stimulated with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) to obtain M1
macrophages and IL-4 or IL-10 to obtain M2 macrophages [143].
2.3.3.3 Macrophage depletion models in mouse
Recent years several DTx-mediated macrophages depletion models have been
developed. Examples are the CD11b-DTRtg
model that depletes CD11b expressing
cells, including macrophages and dendritic cells [144] and the lysM-Cre/DTR model
that depletes wound macrophages [145]. These models show high increase of
neutrophils as a response to the DTx-mediated depletion [144, 145], suggesting
that the models must be used with care to avoid over-interpretation of obtained
results. DTx-mediated depletion models are further discussed in section 2.5.3.3 and
5.1. Macrophages can also be depleted by injection of liposomal clodronate [146].
However, this depletion method targets all phagocytic cells and is therefore not
specific to macrophages.
2.4 Neutrophils
Neutrophils or polymorphonuclear neutrophils (PMNs), are professional
phagocytes and absolutely crucial for the immune defense against invading
microorganisms. PMNs are released from the bone marrow, circulate in blood and
are usually the first leukocytes to be recruited to sites of inflammation. The
number of circulating PMNs differs between humans and mice. In humans, PMNs
correspond to 50–70% of the circulating leukocytes, whereas in mice PMNs only
accounts for 10–25% of the circulating leukocytes [147, 148]. The important role of
PMNs in the immune defense is evident in both humans and mice, as individuals in
total absence of or with significant decreased numbers of PMNs die or suffer from
severe immunodeficiency [149].
13
2.4.1 PMN subtypes and functions
Previously neutrophils have been considered to be a homogeneous cell type
responsible for eliminating invading microorganism. Studies however indicate that
neutrophils might exist in multiple subtypes, with both pro-inflammatory and anti-
inflammatory properties [150-152]. The subtypes likely have diverse roles in
infection, inflammation and cancer immunology. Nevertheless the origin of these
subpopulations needs to be determined, to evaluate whether it is one cell type at
different developmental stages or that has been stimulated differently, or whether
there are truly different neutrophils subtypes [149, 153]. In the following sections,
the focus will be on the role of PMNs during infection.
Upon invasion by pathogenic microorganisms circulating PMNs are recruited to
sites of infection by initially sensing the increased expression of P-selectins and E-
selectins on the surface of the blood vessel endothelial cells. The interaction of P-
selectins and E-selectins with their glycosylated ligands leads to tethering of
neutrophils, followed by rolling, adhesion, crawling, and finally transmigration into
the tissue. Chemokine gradients along the endothelium will guide the PMNs to
preferential sites for transmigration, which occur either paracellular, i.e. between
two endothelial cells or transcellular, i.e. through an endothelial cell. Inside the
tissue, the PMNs follow gradients of chemokines, leading them to the infection site
[149, 153].
PMNs can eliminate pathogens by multiple mechanisms such as phagocytosis,
degranulation, and neutrophil extracellular traps (NETs) [149]. Upon phagocytosis
by PMNs, internalized pathogens are killed through NADPH oxygenase-dependent
mechanisms (reactive oxygen species) or antibacterial proteins, such as cathepsins,
defensins, lactoferrin and lysozyme [149]. The mechanism of phagocytosis is
described in more detail in section 2.1.
PMNs have four different types of granules, or secreted vesicles, that are formed
during their maturation [149]. Each type of granula is filled with anti-microbial
peptides and proteins. The primary azurophilic granules contain myeloperoxidase,
the secondary specific granules contain lactoferrin, the tertiary gelatinase granules
contain matrix metalloproteinase 9 (also known as gelatinase B), and the secretory
granules contain alkaline phosphatase. In contact with pathogens the secretory
vesicles are released first, followed by tertiary, secondary, and primary granules.
PMNs use degranulation to defeat both intracellular and extracellular pathogens by
releasing the granula content either into phagosomes or the external environment
[149].
Highly activated PMNs can also use NETs to eliminate extracellular pathogens. NETs
are composed of the DNA from the PMN itself, to which histones and anti-
14
microbial proteins and enzymes from the granules are attached. The NETs trap the
pathogens, unable them to spread and facilitate internalization by other
phagocytes. In addition, the attached anti-microbial proteins are also thought to
cause direct killing [149].
2.4.2 PMN sensing of pathogens
For a long time PMNs were ignored in the pattern recognition receptor signaling
field, as they were thought to be transcriptionally inactive and limited to respond
to receptor agonists due to their short lifespan. Now it is known that they express a
wide variety of PRRs, including TLRs, C-type lectin receptors, retinoic acid-inducible
gene-like helicase receptors and NLRs. The receptors enable PMNs to sense and
respond to a broad range of microbial and endogenous ligands by changing their
functions and production of cytokines, chemokines and lipid mediators [154].
2.4.3 How to study PMNs?
The high levels of PMNs in blood enable studies of human and mouse PMNs by
isolation of primary cells. Mouse PMNs can also be isolated from bone marrow. The
glycoprotein Ly6G is the best known and most specific marker for PMNs.
2.4.3.1 PMN cell lines
The myeloid leukemia cell line, HL-60, is arrested at the myeloblast differentiation
stage and can be differentiated into PMN-like cells by treatment with various
chemical agents, including retinoic acid and dimethylsulphoxide [155-157].
2.4.3.2 PMN depletion models in mouse
PMNs can be depleted in mice by injection of antibodies. Two monoclonal
depletion antibodies with different specificities are available. α-Ly6G (clone 1A8)
recognize Ly6G and is therefore considered to be specific to PMNs [158]. α-Gr-1
(clone RB6-8C5) mainly detects Ly6G, but also recognizes Ly6C, expressed by PMNs
and monocytes and therefore performs a more unspecific depletion [159, 160].
Due to the higher specificity of α-Ly6G, this antibody is suggested as a better
alternative for PMN depletion than α-Gr-1. PMN-depletion will be further discussed
in section 6.
2.5 Dendritic cells
Roughly defined, DCs are white blood cells that can internalize, process and
present antigens to activate T lymphocytes. However, DCs are so much more than
what this simple definition implies. Since the discovery in 1973 by Ralph Steinman
and Zanvil Cohn many different subtypes and functions have been identified.
Today, DCs are recognized as sentinels of the immune system, as professional
15
antigen-presenting cells and as central actors in the orchestration of the immune
response during tolerance and inflammation [161].
2.5.1 DC subtypes and functions
DCs develop from progenitors in the bone marrow and constitute a very
heterogeneous group of cells with different development, organ localization and
cell surface marker expression, and are therefore complex to categorize. Presently,
DCs can be divided into two major types: conventional DCs (cDCs, also termed
classical DCs) and non-conventional DCs [162]. Non-conventional DCs include
plasmacytoid DCs (pDCs) and monocyte-derived DCs (MoDCs). In addition to cDCs,
pDCs and MoDCs, a DC-like cell type termed follicular DCs belong to this family of
cells [163], although based on appearance rather than function since they are no
“true” DCs and will therefore not be further discussed here.
2.5.1.1 Conventional DCs
The cDCs are characterized as highly phagocytic DCs, specialized in antigen-
processing and antigen-presentation [162]. Briefly, cDCs are subdivided into
migratory and lymphoid-tissue-resident DCs. These subsets can be further divided
based on their surface expression of for example CD4 and CD8. Migratory DCs
include DC subsets in the skin, lungs, intestine, liver, and kidneys. These DCs have
high ability to capture antigens and can migrate from peripheral tissues to
lymphoid organ to present antigens to T lymphocytes and elicit an adaptive
immune response. The lymphoid-tissue-resident DCs reside in the lymphoid organs,
such as lymph nodes, spleen and thymus and lack the ability to migrate [162].
Migration of migratory cDCs to lymphoid organs occurs after capture of antigen
and leads to a mixture of migratory and lymphoid cDCs in the lymphoid tissues
[164].
In the mouse intestine, three different subtypes of DCs have been identified based
on their expression of CD11b and CD8 in combination with CD11c, the pan-DC
marker, and their different functions. CD11b+CD8
- myeloid DCs are located to the
subepithelial dome of the PPs and produce high levels of IL-10 upon stimulation.
These DCs prime T lymphocytes to secrete high levels of IL-4 and IL-10. CD11b-CD8
+
lymphoid DCs are located to the interfollicular regions and produce IL-12p70 upon
stimulation. The double negative population, CD11b-CD8
- DCs are located to both
the subepitheial dome and the interfollicular region and also produce IL-12p70
upon stimulation. Both CD11b-CD8
+ and CD11b
-CD8
- DCs prime T lymphocytes to
produce IFN-γ. [165]
2.5.1.2 Plasmacytoid DCs
pDCs were identified in the 1950s, but first described as plasmacytoid T
lymphocytes and plasmacytoid monocytes based on their plasma cell morphology
16
and expression of either the T lymphocyte marker CD4 or the myeloid cell markers
MHC class II, CD36 and CD68. Not until the 1990s this cell type was characterized as
a dendritic cell [166]. In contrary to the ‘dendritic’ morphology of cDCs, pDCs have
spherical morphology resembling antibody-secreting plasma cells. Regarding
surface markers, pDCs can be distinguished from cDCs by their expression of B220,
Siglec-H, and Bst2 in mice and of BDCA2 (CD303) in humans [167]. pDCs circulate in
the blood stream and lymph vessels and migrate to T lymphocyte areas of
lymphoid organs via high endothelial venules upon stimulation [164]. Due to the
ability of pDCs to produce large amount of type I interferons (IFN) they are
important mediators of antiviral immunity upon viral infection. The IFN production
is induced by sensing of viral nucleic acids through TLR 7 and 9 [166]. In contrast to
cDCs, pDCs are poor T lymphocyte activators. This is due to less efficient capture,
processing and loading of antigens onto MHCII. However, upon activation the
antigen presentation capacity of pDCs increases with increased expression of
MHCII. Because pDCs produce large amounts of IFN and other cytokines, they can
still regulate inflammation and link innate with adaptive immunity by affecting NK-
cells, T lymphocytes, B lymphocytes and other DCs. [168].
2.5.1.3 Monocyte-derived DCs
Monocytes can give rise to DCs in peripheral tissues (e.g. intestine, kidneys, lungs,
and skin) under both steady-state and inflammatory conditions. Such DCs are
called monocyte-derived DCs (MoDCs) and have the ability to internalize antigens
and migrate to draining lymph nodes. [162]. Under inflammatory conditions
monocytes differentiate into MoDCs referred to as inflammatory DCs (iDCs) or TNF-
and iNOS-producing DCs (TipDCs). This development has been observed in infection
models involving bacteria, viruses and parasites [139].
2.5.2 DC sensing of pathogens
The extreme plasticity of DCs enables them to adjust their responses depending on
the nature of the encountered stimulus. DCs have for example been suggested to
be able to distinguish between the presence of an infection contra a cytokine-
mediated inflammation since microbial stimuli, but not inflammatory cytokines,
can stimulate DCs to produce IL-2 [169]. DCs use a broad receptor repertoire to
sense their surroundings and recognize microbial components. The PRRs expressed
by DCs are TLRs, NLRs, RIG-I-like receptors, C-type lectins and mannose receptors
each activated by distinct PAMPs at the cell membrane or within intracellular
vesicles [164, 170]. The binding of a PAMP to a PRR initiates signaling cascades that
eventually result in DC activation and upregulation of MHCII and co-stimulatory
molecules.
17
2.5.3 How to study DCs?
DCs are present in all tissues and in blood. However, the low prevalence makes
isolation procedures inefficient, expensive, and time consuming. DCs constitute
approximately 1% of the total number of leukocytes in PPs and MLN and 2% in
spleen [111]. Alternative methods, such as in vitro differentiation from precursor
cells and depletion models are therefore widely used to study DCs. For cDCs
integrin alpha X chain protein CD11c is the most widely used marker. For intestinal
cDCs the alpha E integrin CD103 is also used.
2.5.3.1 DC cell lines
The best available DC cell line is D1, a growth factor-dependent cell line originating
from C57BL/6 spleen cells through long-term GM-CSF treatment. Unstimulated D1
cells resemble immature DCs, based on the expression of CD11c, MHCII and co-
stimulatory molecules CD80, CD86, and CD40. Stimulation of D1 with
microorganisms, LPS or the cytokines TNFα and IL-1β induce maturation and
upregulation of MHCII, CD86, and CD40 [171].
2.5.3.2 DC differentiation models in mouse and human
Mouse cDCs can easily be differentiated from bone marrow progenitors or
peripheral blood monocytes by stimulation with the growth factor GM-CSF in
absence or presence of IL-4. Mouse pDCs and cDCs can be generated by
stimulation of total bone marrow cultures with Flt3L [172]. GM-CSF differentiated
DCs have been shown to resemble inflammatory DCs and Flt3L differentiated DCs
steady-state resident DCs [173]. From humans, blood monocytes are the most
commonly used precursor cell. Immature myeloid cDCs can be differentiated by
stimulation of blood monocytes with GM-CSF and IL-4. Additional stimulation of
these blood monocytes with transforming growth factor-β (TGF-β) will generate
Langerhans cell-like subsets of DCs [172].
2.5.3.3 DC depletion models in mouse
The CD11c-DTR mouse model is currently the most common model used to study
effects of DC depletion. Mice are insensitive to diphtheria toxin (DTx, an AB toxin)
due to a receptor polymorphism [174]. This was used to construct the CD11c-DTR
mouse, which has a primate DTx receptor (DTR) expressed under the control of the
DC specific CD11c promoter (Itgax) [175]. This set-up allows for conditional knock-
out of cells expressing high levels of CD11c by treatment with DTx [176]. The model
has been quite extensively characterized and a number of problems have been
identified. I) Not all DC subtypes are depleted. Langerhans cells and CD11cint
pDCs
are not affected by the DTx-treatment [177, 178]. This might however be an
advantage if these cells are of interest. II) Other cell types except from DCs are
depleted. The DTx-treatment has been shown to deplete splenic F4/80-CD11c
+
18
marginal zone and metallophilic macrophages [179]. III) The CD11c-DTRtg
mice
cannot be treated with more than two injections of DTx without dying from the
treatment. This results in a limitation of the depletion to four days. The sensitivity
of the CD11c-DTRtg
mice to DTx is thought to be caused by aberrant DTR expression
on non-immune cells, such as gut epithelial cells [175, 179]. Thus, long-term
depletion experiments require radiation chimeras, in which wt mice are
reconstituted with CD11c-DTR bone marrow to construct wt mice with CD11c-DTRtg
hematopoietic stem cells [176]. Two improved CD11c depletion models have been
constructed, the CD11c.DOG and CD11c.LuciDTR [180, 181]. In these models the
DTR has been introduced by a bacterial artificial chromosome and is expressed
under the control of the full CD11c promoter [180, 181]. This is thought to
circumvent aberrant DTR expression on non-immune cells seen in CD11c-DTRtg
mice and makes long-term depletion possible. DTx-mediated depletion models for
specific DC subsets are also available as Langerin.DTR, BDCA2.DTR, SiglecH.DTR,
Clec9a.DTR, and CD205.DTR mice [182]. DTx-mediated depletion will be further
discussed in section 5.1.
Before the introduction of DTx depletion models, injection of liposomal clodronate
has been used to deplete DCs in mice. Clodronate induce suicide by inhibiting the
metabolism of the targeted cells [146]. However, since depletion by clodronate
liposomes requires internalization, this depletion technique is not specific for DC.
Other phagocytic cells, such as macrophages will also be depleted [183]. Depletion
of DCs in mice by injection of depleting antibodies has generally been restrained
because of lack of phenotypic markers specific to individual DC populations and
inefficient susceptibility to antibody-dependent phagocytosis and cytotoxicity in
peripheral tissues. Blood pDCs are an exception as they are efficiently depleted
upon injection of pDCs specific monoclonal antibodies [177].
19
3. Yersinia infection
3.1 Infection route and bacterial dissemination
3.1.1 Enteric Yersinia infection in humans
Enteric Yersinia infections most commonly occur after ingestion of contaminated
food or water. Dairy products, raw meat, and vegetables have been reported as
infection sources [184-186]. Yersinia can replicate and thrive at low temperatures
and thus continue to grow if contaminated food is stored in the refrigerator.
In humans, enteric Yersinia infections often resemble acute appendicitis. Bacteria
are most often found in the mesenteric lymph nodes causing mesenteric
lymphadenitis or localized to the appendix, terminal ileum or ascending colon [187,
188]. Histological examination of infected MLNs and appendices reveal granuloma
formation with central necrosis and microabscess formation [187, 188]. In addition,
ulceration of the intestinal and appendicular mucosa can be observed [188].
Enteric Yersinia infections in humans are usually self-limited and cleared at the
point of MLNs. However, these infections can become persistent and in
immunocompromised patients the bacteria can spread systemically and cause
serious conditions [189-192]. People carrying the HLA-B27 antigen have also been
shown to be extra sensitive to developing reactive arthritis upon enteric Yersinia
infection [193, 194].
3.1.2 Enteric Yersinia infection models in mice
In enteric Yersinia infection models in mice, the infection is initiated through
ingestion of contaminated water or force feeding of concentrated bacterial
suspensions by gavage. In mice, the disease progression is determined by the dose
of infection. In low dose infections, corresponding to 106-10
7 colony forming units
(CFUs) or lower, the infected mice can be divided into three groups depending on if
they clear the infection, develop a persistent or systemic infection (Fahlgren et al.
unpublished data). High dose infection, i.e. 108-10
9 CFUs, results in systemic spread
of the bacteria and subsequent lethal infection of the mice [93, 94, 126, 195, 196].
The susceptibility to Yersinia infection differs between different mouse strains.
BALB/c mice are more susceptible to Yersinia infection than C57BL/6 mice [197,
198]. The higher resistance of C57BL/6 mice is thought to be caused by a more
rapid development of a Yersinia-specific Th1 response and difference in
macrophage activation [198-200], and do not seem to be linked to a particular
gene locus, such as Ity or H-2 as has been reported for the intracellular pathogens
Salmonella, Listeria, and Mycobacteria [197]. In persistent Yersinia infection, FVB/n
mice are the most susceptible, followed by BALB/c and C57BL/6 (Fahlgren et al,
unpublished data).
20
Ingested Yersinia passes through the stomach and initially colonizes the intestine
(Fig. 1). During the first stages of infection, Yersinia enters the Payer’s patches (PPs)
of the small intestine through M cells located in the follicle associated epithelium
[110, 201, 202]. The entry is facilitated by the adhesins expressed by enteric
Yersinia (see section 1.3), together with the phagocytic capacity of the M cells [107,
109]. The invasion of PPs results in formation of a small number of monoclonal
microcolonies, indicating that very few bacteria initially invade the gut epithelium
and establish the infection of the PPs [203]. Early localization of enteric Yersinia in
mouse cecum (corresponding to the human appendix) [67, 196, Paper III], suggests
that bacteria enter the lymphoid follicles of this organ in a similar way as the PPs.
Upon entry into the lymphoid follicles of the intestine, Yersinia encounters the
immune defense (Fig. 1). By the actions of the Yops, which are translocated into
attacking immune cells, Yersinia can avoid elimination by the immune system, stay
extracellular and proliferate in tissues [204]. Proliferation results in an
inflammatory reaction and formation of local microabscesses and ulceration of the
overlying epithelium [204].
Figure 1. Entry and immune cell encounter by Y. pseudotuberculosis in Peyer’s patches and
lymphoid follicles of cecum.
After entry into the lymphoid follicles of the small intestine, Yersinia can
disseminate further to mesenteric lymph nodes (MLNs). This dissemination has
been suggested, but not experimentally proven, to be facilitated by migrating
phagocytic cells [57]. It has also been suggested that bacteria might disseminate
from the PPs not only via the lymphatic system but also by invasion of blood
vessels [110]. In MLN, Y. pseudotuberculosis has been reported to have an
21
extracellular localization nearby neutrophils and macrophages in necrotic areas of
the MLN, and nearby B and T lymphocytes in less inflamed areas [205]. From the
MLN the bacteria can spread to spleen and liver to cause systemic disease
eventually resulting in death of the host. Systemic disease caused by wild-type (wt)
Yersinia usually occurs 3-7 days post oral infection in mice [206, Paper III]. In
similarity to PPs, Yersinia forms monoclonal microcolonies in both spleen and liver
[203] Y. pseudotuberculosis has been shown to disseminate to spleen and liver
already 30 minutes after orogastric inoculation [195]. Noteworthy, the bacteria
involved in this fast systemic infection were also cleared fast and a second
colonization of spleen and liver occurred later. The same study challenges the
believe that enteric Yersinia disseminates systemically via PPs and MLN, by using
signature tagged mutagenesis to show that Y. pseudotuberculosis can disseminate
directly to spleen and liver from a replicating pool in the intestine, without passing
the PPs and MLN. The low number of clones in spleen and liver compared with the
intestine further suggests that a severe bottle-neck limits the systemic
dissemination.
Most of the Yop effector proteins are essential for systemic dissemination of
enteric Yersinia species and many Yop effector mutants are therefore avirulent in
mice (Fig. 2). A mutant lacking the PTPase YopH, is most avirulent and rarely spread
beyond the PPs and the lymphoid follicles of cecum [55, 67, Paper III]. Mutants
lacking either of the GAP YopE, the multifunctional YopM, or the Yop translocation
regulator YopK, are slightly less affected and can spread to the MLN [55, 67, 86, 94,
Paper III]. A Y. enterocolitica yopE mutant is more virulent in mice than a Y.
pseudotuberculosis yopE mutant, which might be due to the presence of YopT,
which can compensate for the loss of YopE activity [55]. Mutants deficient in the
YpkA or the YopJ/P can spread systemically to spleen and liver, but are often
cleared at these locations before them can cause full disease [55, 66, 75]. However,
a Y. pseudotuberculosis yopJ mutant can cause death like the wt strain [66, 79].
Figure 2. Dissemination of wt and yop mutants during oral enteric Yersinia infection in mice
22
3.2 Immune responses during enteric Yersinia infection in mice
Upon enteric Yersinia infection, inflammatory cells are recruited to infected tissues
and a Th1 cytokine response with increased IFN-γ production is initiated [206, 207].
The level of tumor necrosis factor-α (TNF-α) also increases in infected tissues, but
Th2 cytokine (IL-4 and IL-5) levels are low. Thus, indicating that a cellular immune
defense is important against enteric Yersinia species. Pathogenic Yersinia species
have however evolved a number of different strategies to avoid the attack from the
immune defense. These strategies include anti-phagocytosis, inhibition of immune
cell maturation, altered cytokine production, and induction of apoptosis. The
effector Yops are responsible for many of the actions, and they act in synergy to
turn off the immune response.
3.2.1 Yersinia and macrophages
The large distribution of resident macrophages in different tissues results in early
interaction between enteric Yersinia and macrophages in the intestine upon
infection. The infection also leads to increase macrophages levels in infected
tissues [206, 208]. Yersinia targets macrophages through translocation of effector
Yops [111, 209], which have large effects on macrophage functions (Fig. 3). In
macrophages YopH and YopE inhibit phagocytosis [42, 54, 117, 210, Paper I]. Some
Yersinia strains use YopT as an additional anti-phagocytic Yop [54]. Besides
inhibiting phagocytosis, YopH and YopE also inhibit exogenous MHCII antigen
presentation in macrophages. However, macrophages can compensate for this
inhibition by using an autophagy-dependent mechanism instead [211]. YopH also
interfere with the oxidative burst of macrophages [212]. Yersinia inhibits
production of different cytokines in macrophages, e.g. TNF-α, IL-1β through the
action of YopJ and YopE [72, 213, 214]. YopJ/P also induces macrophage apoptosis
[70, 215].
Yersinia infection trigger caspase-1 activation by two different pathways in
macrophages: One pathway responds to the T3SS, and the other pathway is
induced by YopJ [216-219]. The caspase-1 activation (by both YopJ and T3SS) is at
the same time counteracted by YopE, which inhibits caspase-1-mediated
maturation and release of IL-1β through its action on Rac1 [214] and YopM, which
directly inhibit caspase-1 [87]. YopK has also been shown to inhibit inflammasome
activation triggered by the T3SS [220]. Production of IL-1β is one of the most
important inflammatory responses upon infection, since this cytokine is involved in
the regulation of both innate and adaptive immune responses [221].
Studies on macrophages suggest that the immune system can recognize and
distinguish between pathogenic Yersinia and Yersinia lacking a functional T3SS. The
pore-forming proteins YopB and YopD induce NFκB- and type I IFN-regulated genes
in macrophages, independently from TLR, Nod1/2, and caspase-1 [219]. It is
23
suggested that this response informs the host cell of the pathogenic challenge,
leading to a unique response, different from the response to bacteria lacking T3SS.
Moreover, the presence of a functional T3SS during phagocytosis decreases
survival of internalized Yersinia inside macrophages [222, 223]. Consequently, the
T3SS is not only beneficial for Yersinia in the contact with macrophages.
Yersinia is considered to be an extracellular bacterium [204]. In contrast, in vitro
studies with bone marrow-derived macrophages have reported that Y.
pseudotuberculosis can survive inside macrophages and that the survival is
dependent on the response regulator PhoP [224, 225]. These studies however used
bacteria pre-grown at 26 or 28°C, hence without a T3SS primed to translocate Yops
during phagocytosis. We could not confirm these results with a similar
experimental setup, but with bacteria induced for T3SS at 37°C (unpublished data).
Our data further support the observation that plasmid-cured Yersinia survives
better in macrophages than wt [222].
3.2.2 Yersinia and neutrophils
Yersinia infection trigger PMN release into the blood and infiltration of PMNs into
different tissues, such as PPs, MLN and spleen [110, 196, 206, 208, 226, Paper III].
Interestingly, the PMN increase in blood is lower upon infection with avirulent Yop
mutants compared to wt bacteria [208, Paper III]. Thus, indicating that systemic
infection trigger the increased release of PMNs from the bone marrow upon
Yersinia infection.
PMNs are the major target cell for the Yersinia effector Yops [111, 209]. This is
further supported by data showing that depletion of PMNs results in a reduction of
the overall levels of Yop translocation into cells [111]. Inside PMNs the effector
Yops interfere with several important functions (Fig. 3). YopH and YopE are
responsible for inhibiting phagocytosis and oxidative burst by PMNs [54, 227, Paper
I]. During Y. enterocolitica infection YopT plays an additional role in the anti-
phagocytosis against PMNs [54]. Y. pseudotuberculosis can inhibit the formation of
NETs, but which Yop that is responsible for this has not yet been elucidated [94].
Others have reported that Y. enterocolitica (strain E40) expressing YadA, induce
formation of NETs, adhere (presumably to collagen in the NETs) and are killed,
while an YadA-deprived strain only induce the NET formation but do not adhere
and subsequently survives. [228]. In contrast to DCs and macrophages, PMNs are
resistant to YopJ/P-induced apoptosis [229].
The role of PMNs during Yersinia infection has been investigated in several
depletion studies with depletion of Gr-1+ cells, including PMNs and monocytes.
Infection of Gr-1-depleted mice indicates an important role for Gr-1+
cells in the
immune response against enteric Yersinia [230]. Wt bacteria, but also avirulent Y.
24
pseudotuberculosis yopK and Y. pestis yopM mutants become more virulent in mice
after Gr-1-depletion [94, 230-232].
3.2.3 Yersinia and dendritic cells
It is not surprising that Yersinia and other pathogens target DCs, as they play a
central role in the regulation of both innate and adaptive immune responses
against invading microorganisms. DCs are also one of the first obstacles that enteric
Yersinia meets in the intestine (paper III) and must bypass to disseminate
systemically. DCs are targeted in vivo by Y. pseudotuberculosis and Y. enterocolitica
through translocation of the effector Yops [111, 209, 233]. A study on splenic DCs
further show that the DC subtypes are differentially targeted by the Yops, which
are most frequently translocated into CD8+, compared with CD4
+ and CD4
-CD8
- DCs
[233]. Thus, Yersinia infection results in a change in the balance between the DC
subsets in spleen as their antigen uptake and degradation, cytokine production,
and death are differently affected.
Inside DCs the Yops interfere with several important immune functions (Fig. 3).
During Y. enterocolitica infection YopH, YopE, and YopT have been reported to
inhibit bacterial internalization by DCs [53, 234], while during Y. pseudotuberculosis
infection YopE is solely responsible for the anti-phagocytic effect (Paper I). YopJ/P
inhibits DC maturation and T lymphocyte stimulation by blocking antigen uptake
and processing [235, 236], and upregulation of co-stimulatory molecules CD80,
CD83 and CD86, and MHCII [234, 237, Paper I]. Moreover, YopJ/P is responsible for
suppressing the production of several cytokines by DCs, e.g. keratinocyte-derived
chemokine (KC), TNF-α, IL-10, and IL-12 [237], and for inducing apoptosis in DCs
[234, 237] by both caspase-dependent and independent mechanisms [81]. Taken
together, several Yops collaborate to inactivate DCs and prevent their activation of
T lymphocytes.
3.2.4 Yersinia and B and T lymphocytes
By inhibiting antigen presentation and inducing apoptosis of dendritic cells and
macrophages, Yersinia indirectly affects the adaptive immune response. However,
these pathogens have also evolved direct strategies to attack B and T lymphocytes
(Fig. 3). The Yop effector proteins are translocated into B and T lymphocytes, but
less frequently than into the innate immune cells [111, 209]. By the act of YopH,
which dephosphorylates tyrosine-phosphorylated components associated with the
antigen receptor signaling complex, Yersinia directly interfere with B and T
lymphocyte antigen receptor-mediated activation. As a result, B lymphocytes
become unable to up-regulate the co-stimulatory molecule B7.2 in response to
antigen stimulation and T lymphocytes become unable to flux calcium and produce
cytokines [43].
25
Both CD4+ helper and CD8
+ cytotoxic T lymphocytes are involved in the immune
response during enteric Yersinia infection [207, 238, 239]. Increased levels of IFN-γ
and low levels of IL-4 and IL-5, suggest that a Th1 response is initiated [206, 207].
Figure 3. Effects of Yersinia effector Yops on different immune cells.
3.3 Relevance of using mice as a model for human bacterial diseases
Mice are the main experimental model used in immunological studies and their use
have generated a great insight into mechanisms of the immune system [240]. Mice
are widely used experimental models because of their genetic and physiological
similarities to humans, and because their genome can be easily manipulated and
analyzed in various disease and therapy studies. However there are also many
differences between these species, so is it relevant to use this animal as model for
human diseases?
The genome sequencing of mouse and human revealed that both species have
approximately 30,000 genes and that only around 300 genes appear to be unique
to one of the species [241]. Hence, genetically we are more alike than one might
think. However, despite the genetic similarities the immune systems of mice and
humans have evolved differently and even though the overall structure is quite
similar, there are significant differences in both the innate and adaptive immune
response regarding for example development, activation, and response to
challenges [240]. One example is the ratio between lymphocytes and PMNs in the
26
blood. In humans, PMNs compromise the major cell type and accounts for 50-70%
of the total leukocytes in blood, while lymphocytes account for 30-50%. In mice the
ratio is reversed, with 75-90% lymphocytes and 10-25% PMNs [147, 148]. The
functional consequence of this difference is however not clarified.
Recently, a study comparing transcriptional responses to inflammatory conditions
concluded that the genomic responses in mouse models poorly mimic human
inflammatory diseases [242]. The study compared gene expression profiles of
blood leukocytes during different inflammatory conditions, including burn, trauma
and endotoxemia in humans and corresponding mouse models. The results of the
study showed that the transcriptional responses to the different inflammatory
conditions in humans had a higher correlation to each other, than to the
corresponding mouse model and additionally that the responses in mouse models
significantly differed from each other. Thus, underlining that mouse disease models
might poorly reflect the human disease. However, it should be noted that the study
only included one mouse strain and that the human participants were under
medical care, which might have affected the results.
Thus, it is important to consider the limitations of mouse models and caution is
necessary when correlating results from mouse studies to human diseases. Some
important questions to ask are: I) Are mice a natural host of the studied pathogen?
II) Does the disease progress in a similar way and over a similar time course in mice
as in humans? III) Does the mouse model reproduce the major clinical symptoms of
the human disease? IV) Are the same tissues and/or cells affected in the mouse
model as in humans? V) Are the same genes and molecular pathways involved?
[243]
Despite the discrepancies between humans and mice, studies of enteric Yersinia
infections using mouse as virulence model are relevant. The progression of enteric
Yersinia infection in mice resembles yersiniosis in humans in many aspects.
Although not allowing direct correlation to infection in humans, mouse studies help
to identify virulence parameters such as presentation of disease, bacterial loads
and dissemination, extension of tissue damage and to some extent immunological
responses. It is however important to consider the choice of mouse strain, infection
doses and route of infection when interpreting results and relevance for human
pathogenesis. Interpretation of immunological effects also requires a careful
consideration of potential differences between mice and men, which when
possible, can be overcomed by additional in vitro cell culture studies using human
cells.
27
AIMS
The major aim of this thesis was to study the interaction between
Y. pseudotuberculosis and phagocytic cells during early infection. The more specific
aims were:
Investigate the effect of the effector Yop proteins on DC maturation.
Investigate the effect of the anti-phagoctic effectors YopH and YopE on
DCs.
Study the role of DCs during early Y. pseudotuberculosis infection.
Study the role of PMNs during early Y. pseudotuberculosis infection and
for avirulence of yopH and yopE mutants.
28
RESULTS & DISCUSSION
4. PAPER I – Cell type-specific effects of Yersinia pseudotuberculosis virulence effectors
Pathogenic Yersinia species use a T3SS for translocation of a variety of effector
proteins into host immune cells. The actions of the effector proteins against
immune cell functions of macrophages and PMNs have been more extensively
studied than the effects in DCs. In this study we therefore aimed to investigate how
the Yops of Y. pseudotuberculosis affect two key mechanisms of DCs - maturation
and internalization.
4.1 Y. pseudotuberculosis inhibits dendritic cells maturation by the action of YopJ
To investigate the effect of the different Yop effectors on DC maturation, bone
marrow-derived DCs (BM-DCs) were differentiated from mouse bone marrow
progenitors and infected with different Y. pseudotuberculosis strains. Flow
cytometry analysis of the expression of the co-stimulatory molecules CD80, CD83,
and CD86 showed that wt Y. pseudotuberculosis could inhibit DC maturation (Paper
I, Fig. 1C). In contrast, the virulence plasmid-cured strain, which lack the T3SS and
its effector proteins, induced DC maturation. Thus, inhibition of DC maturation was
T3SS dependent. Infection with different single yop mutants revealed that YopJ was
responsible for the inhibition of DC maturation as the yopJ mutant was the only
mutant that induced maturation. Mutants in YopH, YopE and YopM inhibited DC
maturation as efficiently as wt, indicating that these Yop effectors are not involved
in the inhibition of DC maturation. These data confirm previous findings that Y.
pestis and Y. enterocolitica also inhibit DC maturation by the action of YopJ [53,
234, 237, 244, 245]. In conclusion, the human pathogenic species of Yersinia use
the same mechanism to prevent DC maturation.
4.2 Y. pseudotuberculosis resists internalization by dendritic cells through the action of YopE
Internalization is a central defense mechanism employed by immune cells to
eliminate invading microorganisms. Pathogenic Yersinia species can efficiently
avoid internalization and thereby elimination by macrophages and PMNs, which
has been extensively studied in macrophages cell-lines, isolated peritoneal
macrophages, and blood PMNs [42, 117, 210]. At the initiation of this study the
effects of YopH and YopE against DCs were however not elucidated. This together
with the discovery that DCs are one of the major target cells for Yop translocation
29
[246] urged us to investigate the anti-phagocytic capacity of Y. pseudotuberculosis
against DCs.
BM-DCs were differentiated from mouse bone marrow and infected with Y.
pseudotuberculosis wt, virulence plasmid-cured strain, yopH and yopE mutants.
Internalization was evaluated by double immunofluorescent staining, which
discriminates between extra- and intracellular bacteria. Wt bacteria could block
internalization by BM-DCs, while the virulence plasmid-cured strain could not and
was subsequently more frequently internalized (Paper I, Fig. 1D). Interestingly,
infections with the yopH and yopE mutants revealed that YopE is solely responsible
for the anti-phagocytosis against DCs. The internalization assay showed that the
yopH mutant (expressing YopE) could resist internalization to the same level as wt,
while the yopE mutant (expressing YopH) was internalized as the virulence plasmid-
cured strain. Previous data on the anti-phagocytosis against macrophages and
PMNs were also confirmed in bone marrow-derived macrophages (BM-Ms) and
PMNs (BM-PMNs) showing that both YopH and YopE contribute to the anti-
phagocytic capacity of Y. pseudotuberculosis in these cells (Paper I, Fig. 2).
To investigate if this cell type specific feature of Y. pseudotuberculosis was
restricted to DCs from mice or a general effect, the internalization experiments
were performed on DCs derived from human peripheral blood monocytes (PBMC-
DCs) and isolated human blood PMNs (hPMNs). Similar results were seen for the
human DCs and PMNs suggesting that the missing effect in anti-phagocytosis of
YopH against DCs is a general effect (Paper I, Fig. 3B). Further, the data indicate
that YopH is especially important for resisting internalization by more professional
phagocytes, i.e. macrophages and PMNs.
Simultaneously to our study, Adkins et al. investigated the anti-phagocytic capacity
of Y. enterocolitica and concluded that YopH and YopE are both involved in the
inhibition of internalization by DCs for this member of the Yersinia genus [53].
Thus, suggesting that Y. enterocolitica YopH has another role against DCs than Y.
pseudotuberculosis YopH. It should however be noted that this study rather
investigate the internalization by DCs than the anti-phagocytosis by Yersinia since it
analyses percent DCs with intracellular bacteria instead of investigating percent
internalized bacteria, which also consider the ability of the different mutants to
adhere to the cells. Nevertheless, difference in functional importance between
homologous proteins within the Yersinia genus is nothing new. The YopJ protein of
Y. pestis and Y. pseudotuberculosis, and the YopP protein of Y. enterocolitica is one
example. These proteins are differentially expressed and secreted and thereby play
diverse roles during infection with different Yersinia species [76].
30
4.3 The cell specific effect of YopH depends on the mechanism of internalization
The first step in investigating the mechanism behind the cell specificity of YopH was
to analyze the presence of YopH and YopE inside DCs and macrophages. BM-DCs
and BM-Ms were infected with a multiple yop mutant overexpressing either YopH
or YopE. Western blot analysis of intracellular fractions from the two cell types
confirmed that both YopH and YopE were translocated into DCs at similar levels as
into macrophages (Paper I, Fig. 4). Thus, YopH is present inside DCs upon bacterial
contact, but does not contribute to the inhibition of internalization.
The second step in the investigation was to analyze the expression and localization
of YopH target proteins inside DCs. The expression of Fyb and p130Cas was
investigated by Western blot, which revealed that BM-DCs have a markedly lower
expression of both proteins compared with BM-Ms (Paper I, Fig. 5A). However, in
BM-DCs the p130Cas antibody recognized a protein of ~105-110 kDa in size, which
might represent the Cas homologous proteins HEF-1/Cas-L and Sin/Efs [247]. Next,
BM-DCs and BM-Ms were stained to visualize the cellular location of Fyb. In J774
macrophages Fyb is localized to the cell periphery, in areas with high actin
dynamics [248]. In BM-Ms, Fyb showed a similar cellular distribution as in J774
macrophages, with Fyb especially enriched in F-actin rich areas in the cell periphery
(Paper I, Fig. 5B). The immunofluorescence confirmed the low expression of Fyb
detected in BM-DCs by Western blot and further showed that Fyb was diffusely
distributed in the cytoplasm of BM-DCs. Thus, both expression and cellular
localization of the YopH target protein Fyb is strikingly different in BM-DCs
compared with BM-Ms.
The third step in the investigation of the cell type specific effect of YopH was to
analyze the mechanism of internalization used by BM-DCs and BM-Ms. During the
analysis of internalization we observed that the number of attached bacteria and
the number of cells with attached bacteria were significantly higher for BM-Ms
than for BM-DCs. Thus, indicating that macrophages possess a higher capacity for
binding Y. pseudotuberculosis. This was further supported by a kinetic assay
showing that BM-Ms both bind higher number of bacteria but also manage to
internalize them faster than the BM-DCs (Paper I, Fig. 6). While BM-Ms constantly
internalize a high percentage of the attached bacteria, the internalization by BM-
DCs gradually increased over time. β1-integrins on the surface of macrophages and
PMNs mediate the phagocytosis of Y. pseudotuberculosis by binding to the
bacterial surface protein invasin [41]. The internalization of a virulence plasmid-
cured Y. pseudotuberculosis strain lacking invasin by BM-Ms was accordingly
decreased due to affected bacterial binding to the cell (Paper I, Fig. 6). In contrast
the internalization of the same strain by BM-DCs was not affected, showing that
31
the uptake by DCs is independent of the interaction between β1-integrins and
invasin.
Next, live cell microscopy in combination with scanning and transmission electron
microscopy was used to further investigate the mechanism of bacterial
internalization by the two cell types. These advanced microscope techniques
revealed that macrophages mainly internalize Y. pseudotuberculosis through a
zipper-like phagocytosis mechanism resulting in bacteria inside tight phagosomes
(Paper I, Fig. 7A, 7C-G, 8A-B and 8G-H). In contrast, DCs mainly use
micropinocytosis, a random, ruffle-like mechanism resulting in more spacious
phagosomes (Paper I, Fig. 7B, 7H-L, 8E-F and 8I-J). Thus, macrophages and DCs use
different mechanisms to internalize Y. pseudotuberculosis (Fig. 4).
Figure. 4. Y. pseudotuberculosis internalization and anti-phagocytosis against macrophages
and DCs. A) Binding of virulence plasmid-cured Y. pseudotuberculosis (YPIII) to the surface of
a macrophage via invasin-β1-integrin interactions stimulates internalization by a zipper-like
mechanism. A wt bacterium blocks internalization by translocation of anti-phagocytic
effectors YopH and YopE into the macrophage. B) Virulence plasmid-cured Y.
pseudotuberculosis interacting with DCs is internalized by random, ruffle-like
macrophinocytosis. A wt bacterium translocates the anti-phagocytic effectors YopH and
YopE into DCs. Because this internalization mechanism occurs independently of local
32
receptor-mediated responses YopH cannot inhibit internalization. YopE can still influence the
cytoskeletal dynamics and subsequently block internalization.
In summary, these results show that the inability of YopH to inhibit internalization
of Y. pseudotuberculosis by DCs is dependent on the mechanism of internalization.
YopH is translocated into DCs, but since macropinocytosis occurs without the
involvement of β1-integrins and strict receptor mediated signalling mechanisms,
YopH does not have any signalling molecules to act on and cannot prevent
phagocytosis.
33
5. PAPER II – Immune response to diphtheria toxin-mediated depletion complicates the use of the CD11c-DTRtg model for studies of bacterial gastrointestinal infections.
The CD11c-DTRtg
mouse model has been frequently used to investigate the effects
of DC depletion during steady-state and inflammatory conditions [181, 249-251]. In
CD11c-DTRtg
mice, a primate diphtheria toxin receptor (DTR) is expressed under the
control of the CD11c promoter, which allows depletion of CD11chigh
cells by
treatment with diphtheria toxin DTx [175]. In this study we aimed to use the
CD11c-DTRtg
model to investigate the role of DCs during early Y. pseudotuberculosis
infection.
5.1 DTx-mediated depletion of dendritic cells in the CD11c-DTRtg mouse model induces neutrophilia, which results in lower Y. pseudotuberculosis infection
DTx-treated CD11c-DTRtg
mice and PBS-treated wt BALB/c mice were infected with
bioluminescent Y. pseudotuberculosis and the infection was monitored for three
days by in vivo biophotonic imaging using IVIS®Spectrum. Remarkably, Y.
pseudotuberculosis infection of DC-depleted mice resulted in significantly reduced
levels of bacterial colonization at day one and three post infection (p.i.) compared
with undepleted mice (Paper II, Fig. 1). Thus, suggesting that depletion of one of
the most important immune cells might be beneficial for the host and that DCs play
an important role in the establishment of enteric Yersinia infection. However,
analysis of the immune response in uninfected DTx treated CD11c-DTRtg
mice
revealed an induction of the immune response also during steady-state conditions
(Paper II, Fig. 2A-B). The immune response included a massive recruitment of PMNs
to PPs and spleen one to two days after DTx-mediated DC depletion, followed by
an increase of macrophages four days after DTx treatment. This immune response
was not observed in DTx-treated wt mice, showing that the response was not
towards the injected DTx and supporting that the immune response was induced in
response to the DC depletion (Paper II, Fig. 2C). The triggered immune cell
recruitment was also accompanied by increase of KC (homolog to human IL-8) in
uninfected DTx-treated CD11c-DTRtg
mice (Paper II, Fig. 2C). Interestingly, when the
mice were infected by intra-peritoneal injections of bacteria instead of orally, the
observed difference in bacterial colonization between undepleted and DC-depleted
mice was diminished (Paper II, Fig. 3). This result shows that the route of infection
affects the outcome of disease in this model. Further, it indicates that Y.
pseudotuberculosis is more sensitive to the DTx-mediated immune response when
entering via the gastrointestinal route compared with direct systemic spread.
The analyses of the DTx-mediated immune response by immunohistochemistry,
flow cytometry, and a cytokine assay indicated that the response was transient and
34
only triggered by the first DTx-treatment (Paper I, Fig. 2). This means that most of
the PMNs had disappeared at day four after the first DTx-treatment (corresponding
to day three p.i. in infected mice). Hence, to overcome the most activated immune
response, we postponed the onset of infection until three days after the first DTx-
treatment and investigated the bacterial colonization one day p.i.. With the new
experimental setup the difference in bacterial colonization between DC-depleted
and undepleted mice was markedly reduced (Paper II, Fig. 4). Thus, further
supporting that Y. pseudotuberculosis colonization in DC-depleted mice was
restricted as a direct result of the DTx-mediated immune response. Y. enterocolitica
has previously been reported to be restricted to enter into already infected PPs,
presumably due to initiated host immune responses against already invading
bacteria [203]. This is in consistency with our results and might explain why Y.
pseudotuberculosis has colonization problems in DTx-treated CD11c-DTRtg
mice.
CD11c-DTRtg
, CD11c.DOG, and CD11c.LuciDTR mice have all shown increased levels
of neutrophils after DTx-treatment [180, 181, 252] + Paper II), which complicates
the use of this type of depletion model in infection studies (Paper II). Autenrieth et
al. suggest that the observed neutrophilia in DTx-treated CD11c.DOG mice is due to
dysregulation of PMN homeostasis in the absence of DCs [252]. However, the
results in our study do not support this observation. First, PMNs were recruited to
PPs two days after DTx treatment in uninfected CD11c-DTRtg
mice even though
PMNs are usually not present in PPs under steady-state conditions. Secondly, the
increase of PMNs was transient and was not further triggered by the second DTx-
treatment. If DCs would be responsible for regulating PMN homeostasis, PMNs
would be continuously recruited upon DC depletion.
In summary, in addition to the previously reported problems for the CD11c-DTRtg
model regarding for example the limited number of DTx-treatments and depletion
of other cell types, we report that DTx-mediated depletion of DCs induces a
transient PMN response that affects bacterial gastrointestinal infection (Fig. 5). Due
to this response we suggest that infection studies in CD11c-DTRtg
mice and other
DTX-mediated models should not be initiated before the immune status has
returned to normal after the initial DTx-treatment. Consequently, the CD11c-DTRtg
model is best used in the chimeric form, which tolerates repeated DTx-treatments
and thereby long-term depletion. Thus, chimeras can be used in infection studies
where infection is initiated when the immune system has stabilized after depletion.
35
Figure 5. DTx treatment and the subsequent DC depletion results in neutrophilia in CD11c-
DTRtg
mice. The recruited PMNs can eliminate Y. pseudotuberculosis infecting via the oral
route.
36
6. PAPER III – Yersinia pseudotuberculosis efficiently avoids polymorphonuclear neutrophils during early infection
The knowledge about the host cell interactions during the very initial stages of Y.
pseudotuberculosis infection in mice is limited. In this study we therefore aimed to
investigate Yersinia-phagocyte interactions during early infection.
6.1 Y. pseudotuberculosis yopH and yopE mutants, but not the wt strain, interact with PMNs during early infection
Upon entry into the intestinal lymphoid tissues enteric Yersinia encounters
phagocytes. Macrophages and PMNs have been reported to increase at the site of
infection, while the number of DCs are more or less unaffected [110, 111, 196, 206,
208, 226, 253]. In a previous study, we showed that depending on which phagocyte
Y. pseudotuberculosis encounters it needs to be differently equipped to avoid
internalization (Paper I). To avoid phagocytosis by macrophages and PMNs Y.
pseudotuberculosis requires the action of both the PTPase YopH and the GAP YopE,
while YopE alone is enough to withstand internalization by DCs. The importance of
YopH and YopE for avoidance of internalization is also reflected in the virulence of
the corresponding mutants. A yopH mutant is highly avirulent and rarely
disseminates from the intestine, while a yopE mutant can disseminate to the MLN
[55, 67].
To investigate the cell type specific feature of YopH further, we developed an ex
vivo interaction assay in mice. Interaction between Y. pseudotuberculosis and
different phagocytes upon bacterial entry into the lymphoid follicles of the
intestine was investigated by double immunofluorescence using tissue from
infected mice day one and three p.i.. Bioluminescent Y. pseudotuberculosis wt,
yopH and yopE mutant strains were used in the infection, enabling efficient
collection and analysis of PPs and cecums positive for bacteria by in vivo
biophotonic imaging using IVIS®Spectrum. Cecum was finally chosen as the tissue
for analysis, due to a low prevalence of bacteria (especially yopH and yopE
mutants) in PPs at early stages of infection and corresponding results for wt
bacteria in the two tissues (data for PPs not shown). The interaction between single
bacteria and phagocytes in situ was investigated using tissues frozen at day one
and three p.i.. The double immunofluorescence showed that wt Y.
pseudotuberculosis mainly interacted with DCs at day one p.i. (Paper III, Fig. 1A).
The interaction with macrophages was remarkably low and no interaction occurred
with the PMNs recruited to the infected tissue at this time point. At day three p.i.
the level of interaction of wt bacteria with DCs and macrophages resembled day
one p.i. (Paper III, Fig. 1B). However, at this stage wt bacteria also interacted with
the massive number of PMNs recruited to the infected tissue. Interestingly,
infection with the virulence deficient yopH and yopE mutants resulted in a
37
strikingly different pattern of bacteria-phagocyte interaction. At day one p.i. the
level of interaction with DCs was similar for yopH and yopE mutants, as that
observed for the wt. However, in contrast to wt bacteria the mutants also
interacted with PMNs at this early time point. At day three p.i. interaction of the
mutants with both DCs and PMNs were more frequently occurring than for wt
bacteria. Taken together the results show that yopH and yopE mutants interact
with PMNs in the intestine both earlier and more frequently than wt. This suggests
that wt bacteria can avoid interactions with PMNs by the action of these Yop
effectors.
6.2 PMN depletion increase the virulence of Y. pseudotuberculosis
To further investigate the role of PMNs during initial intestinal infection and the
way in which Y. pseudotuberculosis avoid these cells, we employed a PMN
depletion model. Mice were depleted of PMNs by intraperitoneal injection of α-
Ly6G antibodies and infected with bioluminescent Y. pseudotuberculosis wt, yopH,
yopE, and yopK mutants. The yopK mutant was included as a control in this
experiment, as the virulence of this mutant has previously been shown to increase
in the absence of PMNs [94]. The infection was monitored by physical
examinations, flow cytometry, and in vivo biophotonic imaging of whole animals
and organs using IVIS®Spectrum.
The physical examination of the mice included visual observation of disease
symptoms such as ruffled fur, diarrhea, hunchback posture, and inactivity. Disease
symptoms were initially observed at day three p.i.. At this time point PMN-
depleted mice infected with wt, yopE, and yopK mutants showed slightly more
symptoms compared with the undepleted mice (Paper III, Table 1). At termination
of the infection at day five p.i. the yopE mutant infected PMN-depleted mice still
had more severe symptoms of disease compared with the undepleted mice. Wt
and yopK mutant infected PMN-depleted mice showed that same level of
symptoms as the undepleted mice at day five p.i.. Undepleted and PMN-depleted
mice infected with yopH mutant did not show any symptoms of disease. Thus, the
physical examinations of the mice indicated that PMN-depleted mice infected with
wt, yopE or yopK mutant were initially more affected by disease than infected
undepleted mice.
To monitor PMN depletion, blood samples were collected and analyzed by flow
cytometry for cellular expression of the PMN marker Gr-1 (Ly6G/6C) and major
histocompatibility complex (MHCII). Uninfected, undepleted mice had stable levels
of Gr-1high
MHCII- cells (corresponding to PMNs) (Paper III, Fig. 2B). In wt infected
undepleted mice the level of Gr-1high
MHCII- cells gradually increased in the blood
during the infection. Interestingly, flow cytometry analysis of blood samples from
mice infected with yopH, yopE and yopK mutants revealed that these mutants
38
induced a significantly lower release of PMNs into the blood circulation compared
with wt (Paper III, Fig. S1). This finding indicates that release of PMNs is triggered
by systemic dissemination of this bacterium, which is in consistency with previous
reported data [208]. This also explains the gradually increasing levels of PMNs in
blood upon wt infection. In our study this conclusion is further supported by the
increase of PMNs in blood from one of the yopH mutant infected mice. This
particular mouse had an unusually observed, but constrained bacterial plaque in
the liver at day five p.i. and subsequently also higher increase of PMNs in blood
compared with the remaining yopH mutant infected mice. All α-Ly6G-treated mice
showed very low levels of Gr-1high
MHCII- cells in the blood (Paper III, Fig. 2B). Thus,
confirming that the α-Ly6G antibodies were injected in sufficient amounts for
depletion of PMNs also during Y. pseudotuberculosis infection.
To monitor infection progression the infected mice were anesthetized and signals
from the bioluminescent bacteria were measured by IVIS. Mice were also dissected
to investigate the bacterial dissemination in organs by IVIS. The external IVIS
analysis indicated that the colonization efficiency of all bacterial strains increased
in the absence of PMNs at day one p.i. (Fig. 6 and Paper III, Fig. 3 and Fig. 4). For
the wt strain the increased colonization efficiency was seen as significantly higher
external bioluminescent signals in PMN-depleted mice compared with undepleted
mice. The frequency of bacterial dissemination to MLN was not affected by PMN
depletion thus indicating that wt bacteria can efficiently resist the PMN-mediated
defense in the lymphoid follicles of the intestine. This was also in consistency with
the interaction data for the wt strain (Paper III, Fig. 1). For the yopH, yopE, and
yopK mutants both increased external bioluminescent signals and increase
bacterial dissemination to MLN were observed in PMN-depleted compared with
undepleted mice. Thus, indicating that PMNs are responsible for restricting these
mutants from dissemination to the MLN.
Previous studies in PMN-depleted mice show that Y. pseudotuberculosis yopK and
Y. pestis yopM mutants become more infectious in the absence of PMNs [94, 231,
232]. Taken together, our and others data show that many Yops contribute to the
PMN-resistant phenotype of Yersinia species and are important for the efficient
PMN escape of wt bacteria. A fully equipped Yersinia is not markedly affected by
the anti-microbial actions of PMNs. Hence, PMN depletion only accelerates the
infection progression slightly. For the avirulent Yop mutants the observed result of
PMN depletion is more pronounced as the absence of PMNs enable these bacteria
to disseminate faster and reach deeper tissues.
39
Figure 6. PMN depletion by injection of α-Ly6G antibodies results in increase initial
colonization by Y. pseudotuberculosis wt bacteria and faster dissemination of avirulent yopH,
yopE and yopK mutants to mesenteric lymph node.
6.3 Undepleted immature PMNs compensate for the loss of mature PMNs in PMN-depleted mice
At day three and five p.i. the physical examination of disease symptoms, and the
IVIS analysis of external bacteria signals and bacterial dissemination in organs
revealed that a difference in the infection progression could no longer be observed
between undepleted and PMN-depleted mice (Paper III, Table 1, Fig. 3 and data not
shown). Thus, suggesting that a compensatory immune response is initiated in the
absence of PMNs. This was confirmed by the flow cytometry analysis performed to
monitor the PMN depletion, which revealed that a Gr-1int
MHCII- cell population
increased in a similar pattern in PMN-depleted mice, as Gr-1high
MHCII- cells
increased in undepleted mice (Paper III, Fig. 2 and Fig. 5A). Further analysis by flow
cytometry showed that the Gr-1int
MHCII- cell population in infected PMN-depleted
mice was mainly immature PMNs (Gr-1int
F4/80-), but also inflammatory monocytes
(Gr-1int
F4/80+) (Paper III, Fig. 5B). Immunohistochemistry showed that these
immature PMNs behaved like mature PMNs and were also recruited to infected
tissues (Paper III, Fig. S2). Flow cytometry also revealed a slight increase of Gr-
1+F4/80
+ monocytes in blood of infected PMN-depleted mice, which was
accompanied by an increase of Ly6C+F4/80
+ inflammatory macrophages in infected
tissues of PMN-depleted mice shown by immunohistochemistry and
immunofluorescence (Paper III, Fig. 5A, Fig. S2, and Fig. S3). Similar results
regarding immature PMNs and macrophages have been observed in the oviducts of
PMN-depleted mice infected with Chlamydia [254]. Taken together these findings
suggest that release of immature PMNs is a general effect in α-Ly6G-treated mice
upon infection and that the expression of Ly6G on these cells is not sufficient for
antibody depletion.
The incomplete depletion using α-Ly6G prevents the ability to investigate whether
the previously observed effects of increased Yersinia virulence in Gr-1-depleted
40
mice are dependent on PMNs or PMNs in combination with inflammatory
monocytes. However, the increase of inflammatory monocytes and inflammatory
macrophages in PMN-depleted mice in this study indicate that that these cells play
a role during Yersinia infection. Thus, suggesting that mice treated with α-Gr-1 will
be more affected by Yersinia infection than mice treated with α-Ly6G.
41
CONCLUSIONS
PAPER I
Y. pseudotuberculosis inhibits DC maturation through the action of YopJ.
Y. pseudotuberculosis avoids internalization by macrophages and PMNs
through the action of YopH and YopE, while YopE alone can inhibit
internalization by DCs.
The cell type specific feature of YopH is dependent on the different
mechanisms of internalization used by macrophages and DCs.
PAPER II
The CD11c-DTRtg
model is complicated to use for infectious studies due to
DTx-mediated neutrophilia.
The induced immune response of CD11c-DTRtg
mice and other DTx-
mediated depletion models upon DTx-treatment must be carefully
investigated and taken into consideration before initiation of infection to
avoid misinterpretation of obtained results.
PAPER III
Avoiding interaction with PMNs is a key feature of pathogenic Yersinia,
which allows colonization and effective dissemination in mice.
Y. pseudotuberculosis yopH and yopE mutants, but not the wt strain,
interact with PMNs in the lymphoid follicles of the intestine during the
initial stage of oral infection.
PMN-depletion results in more efficient initial colonization by the wt strain,
as well as yopH, yopE, and yopK mutants. In addition, dissemination to MLN
of the virulence attenuated mutants increases in absence of PMNs.
PMN-depletion with α-Ly6G antibody is problematic since immature PMNs
are not depleted and can compensate for the loss of mature PMNs in the
immune response towards invading bacteria.
42
ACKNOWLEDGEMENTS
At the age of fourteen me and my friends were asked about our ambitions in life.
My friends did not have a straight answer, but I immediately said “I am going to be
a scientist”. Wherever my heart will finally lead me in life, I at least did science for 6
years and 4 months (project student + PhD). This time has literary been filled with
blood, sweat and tears, but most of the time a lot of fun and fantastic people.
Thank you Maria for accepting me as your PhD student and making my teenage
dream come true. Thank you for being enthusiastic, for challenging me with
gradually increasing responsibility, for sending me to interesting conferences all
over the world and for cheering me up when my mouse experiments showed
totally unexpected and strange results. Those **** neutrophils! I will never forget
the time I spent in your lab.
My fellow group members during the PhD: Anna, my Baloo and second supervisor
who has taken care of me from day one and taught me (almost) all the importat lab
knowledge I have - From how to infect a mouse to how to make noodles in the
microwave oven. Without your good advices about experiments, writing, and life in
general, this journey would not have been the same. It has been truly fantastic
working with you! Sara, the lab was never the same after you left. I missed our
talks about everything and nothing, you singing in the lab, and even your stuff
taking over my lab bench and your phone ringing in the office when you were not
there. Thank you for accepting to be my toastmaster. Good luck with the “football
team”! Kemal, I am gladly handing over the important job of being the senior PhD
student in the group to you. Thank you for saving me when I locked myself out of
my apartment, twice. Kristina, thank you for supplying me with the pIB30 mutant
and lux-strains, for help during the stressful PMN depletion experiment and for
apples and yarn. Saskia, thank you for interesting talks, leftover PMNs, tasty fika
and a lot of nice chocolate. Nayyer, I really like your “I will kill you!” and “Are you
kidding?”-comments. Never stop using them! Take good care of Anna when I am
gone. Karen, for contributions to my first publication. To my hardworking students,
Hanna, Tönis and Anna vdM. Thank you for doing experiments for me! I hope I did
not scare you, but inspire you as my first supervisors, Gosia and Ingrid, did.
6 years and 4 months is a long time and it makes me sad to leave the Department
of Molecular Biology and all its fantastic people. Thank you group HWW, MFr, ÅF
DMi and VS for interesting journal clubs and project presentations. Maybe I will
come back and terrorize you with more immune response papers and talks in the
future. Thank you to the people in the bunker and at the ”PhD-table” for
company, interesting talks, jokes, and fika through all these years. Thank you to my
partners in crime during the years in the marketing group, during teaching and to
43
all the people making crafting nights and molbiol running competitions possible. An
extra thank you to Mari for fun activities in- and outside work. Let us continue
dreaming of a postdoc in the States! Maria N for pep talks and for saving my ass
during the depletion experiments. To Jenny for great company during the trip to
Athens and course at Spetses. To Rutan for help with FACS and for letting me use
your machine. To Marie A, Bhupender and Ingrid for help with the microscopes. To
Johnny for many acute orderings of antibodies and liquid nitrogen, and for lending
me tools, which I always returned! To Ethel for all paperwork and other important
things, and the media ladies for providing me with endless numbers of plates,
buffers and media. Thank you Marek for computer support and Marianella for
keeping the department cleaner than everyone else. And finally, Sven for being the
funniest boss ever!
My friends during the undergraduate student time and after, Sofia, Reine,
Annasara, Annika, Marie and John. Thank you for dinners and other fun get
togethers. And thank you for continuously inviting me to birthday celebrations
even though I am always busy with something else. Anna, maybe you were the
most cleaver in the class. The one who finished, but decided to work with
something totally different. One thing is at least true - Malmö is too far away!
My family, relatives and friends, this thesis is for you! Now, when it is finally time
for me to move on, you might actually understand what I have done during all
these years. Thank you for being interested, asking questions and worrying about
me during periods of too much work. Thank you for lighting up my life outside
work! And a million thanks to those of you who have helped me take care of
Hedvig. This work would not have been finished in time without you! Mom and
Dad, I obviously would not be where I am today without you. Thank you for the
genetic and social contributions that have made me the person I am, and thanks
for letting me be me. Hedvig, my sweet little Bolonka. The best friend anyone can
have! No one will ever be as happy as you are to see me, independently if I left you
for ten minutes or a month. Thank you for all the unconditioned love, joy and
happiness you give me!
Where ever my heart will lead me now, I am grateful that all of you passed me by!
44
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