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Modulation of cellular innate immune responses by lactobacilli
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- Pippi Långstrump
Örebro Studies in Life Science 10
MATTIAS KARLSSON
Modulation of cellular innate immune responses
by lactobacilli
© Mattias Karlsson, 2012
Title: Modulation of cellular innate immune responses by lactobacilli.
Publisher: Örebro University 2012
www.publications.oru.se
[email protected]
Print: Örebro University, Repro 05/2012
ISSN 1653-3100
ISBN 978-91-7668-872-4
Abstract
Mattias Karlsson (2012): Modulation of cellular innate immune responses
by lactobacilli. Örebro Studies in Life Science 10, 84 pp.
Lactobacillus is a genus of lactic acid bacteria frequently used as healthpromoting probiotics. Using probiotics to treat or prevent infections is a
novel experimental approach with vast impact on future therapy. Lactobacillus rhamnosus GR-1 is a probiotic investigated for its ability to reduce
urogenital disease including urinary tract infections caused by pathogenic
Escherichia coli. L. rhamnosus GR-1 has been shown to modulate immunity, thought to influence its probiotic effect. In this thesis, the aim was to
study immunomodulation by L. rhamnosus GR-1 and other lactobacilli,
with emphasis on elicited immune responses such as nuclear factor-kappaB
(NF-κB) activation and cytokine release from human urothelial cells.
Viable, heat-killed, and isolated released products from L. rhamnosus
GR-1 augmented NF-κB activation in E. coli-challenged urothelial cells.
Blocking of lipopolysaccharide binding to toll-like receptor 4 completely
quelled this augmentation. Size-fractionation, urothelial cell challenge, and
two-dimensional gel electrophoresis of L. rhamnosus GR-1 released products presented several candidate proteins with NF-κB modulatory actions
including chaperonin GroEL, elongation factur Tu, and a protein from the
NLP/P60 protein family. While tumor necrosis factor was correspondingly
augmented by L. rhamnosus GR-1, the release of two other cytokines,
interleukin (IL)-6 and CXCL8, was reduced. Similar effects were observed
in macrophage-like cells stimulated with L. rhamnosus GR-1.
Many immunomodulatory effects of lactobacilli are believed to be species and strain dependent. Therefore, twelve Lactobacillus strains were
used to screen for their effects on CXCL8 release from urothelial cells. A
majority of these strains were able to influence CXCL8 release from the
cells. Phylogenetic analysis revealed close evolutionary linkage between
lactobacilli with similar actions on CXCL8. Increased knowledge on probiotic bacterial products and the mechanism(s) of action could lead to improved future treatments for infections.
Keywords: cytokines, immunomodulation, lactobacilli, probiotics, urinary
tract infections, urothelium.
Mattias Karlsson, School of Science and Technology
Örebro University, SE-701 82 Örebro, Sweden, [email protected]
List of studies
This thesis is based on the following studies, referred to in the text by their
roman numerals (I---V).
Study I
Released substances from lactobacilli influence immune
responses in human epithelial cells.
Mattias Karlsson, Simon Lam, Nikolai Scherbak, and Jana Jass
(2010) In vivo: 24: 367-368 (In: Abstracts of the 3rd SwedishHellenic life sciences research conference, Athens, March 25-27,
2010)
Study II
Lactobacillus rhamnosus GR-1 enhances NF-kappaB
activation in Escherichia coli-stimulated urinary bladder
cells through TLR4.
Mattias Karlsson, Nikolai Scherbak, Gregor Reid, and Jana Jass
(2012) BMC Microbiology 12:15 (doi:10.1186/1471-2180-12-15)
Study III
Substances
released
from
probiotic
Lactobacillus
rhamnosus GR-1 potentiate NF-κB activity in Escherichia
coli-stimulated urinary bladder cells.
Mattias Karlsson, Nikolai Scherbak, Hazem Khalaf, Per-Erik
Olsson, and Jana Jass
(unpublished study)
Study IV
Probiotic Lactobacillus rhamnosus alters inflammatory
responses of bladder epithelial and macrophage-like cells in
co-culture
Hanan Abuabaid, Mattias Karlsson, Nikolai Scherbak, Per-Erik
Olsson, and Jana Jass
(unpublished study)
Study V
Lactobacilli differently regulate expression and secretion of
CXCL8 in urothelial cells.
Mattias Karlsson and Jana Jass
(accepted for publication in Beneficial Microbes)
Published studies are reproduced with permission from the copyright holders.
Main abbreviations
ATCC
BV
DNA
EF-Tu
EFSA
ELISA
FAO
FITC
GAPDH
GCSF
HIV
HRP
HMP
Ig
IL
kDa
L.
LPS
LTA
MAP
MetaHIT
mRNA
NF-κB
NLR
PAMP
PBS
PMA
PMB
PRR
qPCR
TCP
TLR
TNF
UPEC
UTI
WHO
American Type Culture Collection
Bacterial vaginosis
Deoxyribonucleic acid
Elongation factor Thermo unstable
European Food SafetyAuthority
Enzyme-linked immunosorbent assay
Food and Agriculture Organization (United Nations)
Fluorescein isothiocyanate
Glyceraldehyde-3-phosphate dehydrogenase
Granulocyte colony-stimulating factor
Human immunodeficiency virus
Horseradish peroxidase
Human Microbiome Project
Immunoglobulin
Interleukin
Kilodalton
Lactobacillus
Lipopolysaccharides
Lipoteichoic acid
Mitogen-activated protein
Metagenomics of the Human Intestinal Tract
Messenger ribonucleic acid
Nuclear factor-kappa B
Nucleotide oligomerisation domain-like receptors
Pathogen-associated molecular pattern
Phosphate buffered saline
Phorbol 12-myristate 13-acetate
Polymyxin B
Pattern recognition receptor
Quantitative polymerase chain reaction
TIR-containing protein
Toll-like receptor
Tumor necrosis factor
Uropathogenic Escherichia coli
Urinary tract infection
World Health Organization
Table of Contents
INTRODUCTION .................................................................................. 13 Aims ........................................................................................................ 14 THE IMMUNE SYSTEM ........................................................................ 15 Innate immunity ....................................................................................... 15 Mucosal responses ................................................................................... 19 Responses towards microorganisms and tolerance ................................... 20 THE HUMAN MICROBIOTA ............................................................... 21 The importance of microbes ..................................................................... 21 Vaginal lactobacilli and health ................................................................. 22 PROBIOTICS .......................................................................................... 25 Immunomodulation by probiotics ............................................................ 26 Additional probiotic effects ...................................................................... 28 Safety issues ............................................................................................. 28 URINARY TRACT INFECTIONS ......................................................... 31 UTI pathogenesis ..................................................................................... 31 Urogenital probiotics ............................................................................... 34 METHODOLOGY .................................................................................. 37 Cell challenges ......................................................................................... 37 Culture of cells and bacteria ................................................................. 37 Isolation and fractionation of released products from lactobacilli ........ 38 Co-culture of urothelial cells and macrophage-like cells ....................... 38 Detection of immunological outcomes ..................................................... 39 NF-κB activation (luciferase assay) ....................................................... 40 Quantitative PCR ................................................................................. 41 Fluorescence microscopy and native immunoblots ............................... 41 ELISA .................................................................................................. 42 RESULTS AND DISCUSSION ................................................................ 43 The urogenital probiotic L. rhamnosus GR-1 influences
tissue cell responses to E. coli ................................................................... 44 L. rhamnosus GR-1 modulates E. coli recognition in tissue cells .............. 47 Released products from L. rhamnosus GR-1 are responsible for NF-κB
augmentation and contain putative immunomodulatory substances......... 49 The effects of L. rhamnosus GR-1 on immune cells are complex ............. 52 Lactobacilli demonstrate inter-species variability in
urothelial cell CXCL8 modulation ........................................................... 55 CONCLUSIONS AND FUTURE PERSPECTIVES .................................59 Conclusions.............................................................................................. 59 Future perspectives ................................................................................... 59 ACKNOWLEDGEMENTS ......................................................................61 REFERENCES .........................................................................................63 SVENSK SAMMANFATTNING.............................................................79 Inledning .................................................................................................. 79 Förstärkta immunsvar av L. rhamnosus GR-1.......................................... 80 Sekreterade substanser bidrar aktivt till förändrade immunsvar ............... 81 Studier på vita blodkroppar ger mer information ..................................... 81 Stora variationer mellan laktobaciller....................................................... 82 Nya möjligheter ....................................................................................... 83 INTRODUCTION
Hippocrates, by many regarded as the father of medicine, supposedly said
“let food be your medicine and medicine be your food” [1]. For someone
like me, that has been studying the effects of lactobacilli on human cells,
such a statement comes with a highly personal interpretation. According to
Hippocrates, food and medicine is one and the same and used to maintain
health. A modern view on such a piece of (functional) food is – that it in
addition to being nutritious and free from various toxic compounds – is
also rich in health promoting microorganisms.
In almost every shop and pharmacy today, products have an addition of
allegedly health-promoting lactic acid bacteria. These microorganisms have
been given the epithet “probiotics”, meaning “for life” and is an attractive
antonym to the familiar antibiotic drugs (against life). The positive health
benefits of consuming these bacteria are now well established, used for
treating a dysfunctional bowel as well as a possible treatment of cancer [2].
No other single drug can compete with such qualities. Maybe it’s because
of those unique and multiple talents that these microbes have become such
an attractive target for charlatans. Sadly, there are a notable number of
“probiotic” products available, declaring they can make your life better in
an uncountable number of ways. Apart from maintaining regularity, they
can also work as antiperspirants or make your hair grow back. That is
what they claim of course. In real life, many of these products don’t even
contain microorganisms with documented health benefits or in some cases
– they don’t have any bacteria at all in them! Luckily, there is ample
evidence for using probiotics, in cases where such an epithet is justified.
During the last ten years, there has been an increased scientific interest
in what probiotic bacteria can do for our health and how they go about it.
The mechanisms underlying probiotic actions are largely unknown
although their impact on host responses including immune regulation is
thought to be a key factor. Cohesive studies describing the use, functions,
mechanisms and eventual side effects of probiotic bacteria are needed in
order to increase the currently limited body of knowledge. Many researchers aim at answering questions such as how these bacteria can make a dysfunctional bowel behave normal after only a few weeks of probiotic
regimen, or how probiotics can decrease the risk of respiratory infections
although they were consumed orally without colonising the airway
epithelium. There are many questions that need to be answered, especially
those concerning reactions on the cellular level. By recognising the many
entities governing probiotic-maintained health – commensal microorganisms, probiotics, pathogens, and the host – we realise that studies on
MATTIAS KARLSSON I
13
probiotics need to be well coordinated and comprise many aspects of
microbiology, physiology, and immunology.
The studies on which this thesis is based deals with the elicited cellular
responses by human cells that face probiotic microbes. The emphasis is on
responses from pathogen-challenged epithelial cells originating from the
urinary system, although some results on how professional immune cells
behave after Lactobacillus treatment have also been included. Although I
have gained a lot of knowledge on probiotics in general and elicited
reactions of urinary epithelial cells more specifically during my time as a
doctoral candidate, I have first and foremost learned that trying to answer
scientific questions raises an equal or greater number of new ones.
Surprisingly, I have also realised that our modern beliefs on health are not
that different from that of an ancient healer and philosopher such as
Hippocrates.
Aims
The work behind this thesis has been guided by specific objectives, listed
below.
• Analysing
urogenital
probiotic
lactobacilli
(Lactobacillus
rhamnosus GR-1 and L. reuteri RC-14) influence on urinary epithelial cell immunity.
• Isolating and characterising immunomodulatory factors from
L. rhamnosus GR-1.
• Screening for immunomodulatory abilities within the Lactobacillus
genus, exposing differences on species and strain level.
• Identifying the immunomodulatory impact of L. rhamnosus GR-1
on macrophages.
14
I MATTIAS KARLSSON THE IMMUNE SYSTEM
The eukaryotic immune system is a fascinating creation. Through evolution
it has developed from a few germline-encoded proteins into a complex
network of receptors, differentiated cell types, and antibody production. Its
main role is to help protect the host from unwanted colonisation by pathogenic organisms such as bacteria. In higher animals, the immune system
can be divided into two arms: the innate and adaptive immune system.
While innate immunity is a conserved reaction to invariant non-self
products, adaptive immunity is based on expansion of B-lymphocyte and
T-lymphocyte subsets leading to antibody production, macrophage
activation, or direct cytotoxicity. Both arms of the immune system are
important for controlling the growth of various pathogens yet at the same
time discriminate between harmless and harmful organisms, as well as
other substances presented to the body.
Innate immunity
The first immunological reaction to microbes entering the body is in large
governed by germline-encoded pattern recognition receptors (PRRs) that
are found on the surface of eukaryotic cells. Once these PRRs are properly
attached to their cognate ligands (primarily microbial products), the cells
initiate the production of substances that increase cellular stability, induce
inflammation, and promote elimination of invading pathogens.
Many PRRs are part of a major family known as toll-like receptors
(TLRs) present in multicellular organisms [3]. They are predominantly
transmembrane proteins found on professional immune cells such as
macrophages. These proteins have an extracellular portion able to bind
certain invariant structures with high affinity, while the intracellular
domains carry out effector functions. The bacterial TLR ligands have
collectively been termed pathogen-associated molecular patterns (PAMPs)
since they were first identified when studying invading pathogens. Today,
numerous TLRs have been described in mammals; humans have at least ten
and mice eleven characterised receptors where each receptor has its specific
subset of ligands (Table 1).
Although TLRs are part of the same protein family, they exhibit some
differences, specifically ligand binding and subcellular localisation. While
most TLRs are found on the surface of eukaryotic cells, a few reside within
the cytosol. Their localisation can generally be explained by their respective
PAMP specificity: transmembrane TLRs bind motifs found on extracellular
pathogens, whereas cytosolic TLRs preferentially identify genetic
components (DNA and RNA) as a means of finding viruses and bacteria
MATTIAS KARLSSON I
15
present in the cytosolic compartment. Recently, a new family of PRR
proteins has been identified, with so far only cytosolic members. These
nucleotide oligomerisation domain-like receptors (NLRs) bind cell wall
residues from both Gram-positive and Gram-negative bacteria and their
DNA, as well as toxins, and have proven important in defending the cell
from intracellular pathogenic bacteria [4].
Table 1. Mammalian TLRs and important ligands.
Receptor and ligand
Studied organism
Reference
Mouse
[5]
Lipoproteins
Human
[6]
Lipoteichoic acid
Human
[7]
Yeast zymosan
Mouse
[8]
Human, mouse
[9]
Lipopolysaccharides
Human
[10]
Heat-shock proteins
Human, mouse
[11, 12]
Murine β-defensin
Mouse
[13]
Mouse, chinese hamster
[14]
Mouse, chinese hamster
[15]
Imidazoquinolines
Human, mouse
[16]
Single-stranded RNA
Human
[17]
Human
[17]
Human
[18]
Mouse
[19]
TLR1
Lipoproteins (together with TLR2)
TLR2
TLR3
Double-stranded RNA
TLR4
TLR5
Flagellin
TLR6
Peptidoglycan (together with TLR2)
TLR7
TLR8
Single-stranded RNA
TLR9
Bacterial CpG motif
TLR10
No known ligands
TLR11 (only in mouse)
Profilin
16
I MATTIAS KARLSSON TLR4 was the first human member of the TLR protein family to be identified [20]. Since its discovery in 1997, this receptor has served as a general
model for the intracellular signalling pathways ensuing TLR activation.
Natural, high-affinity ligands for TLR4 are lipopolysaccharides (LPS),
substances found exclusively in the cell wall of Gram-negative prokaryotes,
yet absent in animal cells [10]. The recognition of LPS by TLR4 is a good
illustration of how multicellular eukaryotes discriminate between “self”
and “non-self”. Since LPS is not present anywhere on animal cells, all LPS
molecules in an animal must be due to microbial presence, an event that
might become a threat to the individual organism. One example of
microbes that can activate TLR4 is the coliform bacterium Escherichia coli,
primarily because of its outer cell envelope rich in LPS. Once in contact
with TLR4 on the surface of eukaryotic cells, the LPS molecule acts as an
agonist – triggering the intracellular pathways that lead to a number of
changes that eventually lead to transcription of genes essential in pathogen
removal.
Through extensive research, scientists have been able to map the very
complex signalling cascades executed after agonist binding. In short, TLR
activation affects two large pathways inside the cell: the mitogen-activated
protein (MAP) kinase and nuclear factor-kappa B (NF-κB) pathway
(summarised in Figure 1) [3]. MAP kinase activation is basically a series of
phosphorylation events that lead to the expression of certain genes
involved in a wide range of cellular decisions from mitosis to immune
activation [21]. NF-κB was initially identified as an important regulator in
the development of antibody-producing B-lymphocytes, although its role in
other immunological processes was soon thereafter determined. Activation
of NF-κB is primarily executed by releasing NF-κB dimers from inhibitory
proteins that keep the transcription factor inactive within the cytosol. Once
these inhibitory factors have detached, NF-κB can migrate to the nucleus
and induce gene expression by binding to short sequences located upstream
of inducible genes [22].
Genes regulated by MAP kinases and NF-κB dimers are numerous,
although many of them are important in the immunological processes that
eventually culminate in pathogen clearance. One of the most important
groups of proteins produced during immune activation are the cytokines,
small proteins released by cells that help shape immune responses.
MATTIAS KARLSSON I
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Figure 1. Simplified overview of TLR activation leading to transcription of NF-κBand MAP kinase-regulated genes. DD, death domain; NF-κB, nuclear factor-κB;
IκB, inhibitor of κB; IκK, IκB kinase; IRAKs, IL-1 receptor- associated kinases;
JNK, c-Jun-N-terminal kinase; MAPK, mitogen-activated protein kinases; MKK6,
MAP kinase kinase 6; MyD88, myeloid differentiation factor 88; NLS, nuclear
localisation signal; P, phosphorylation; p38, p38 MAP kinases; TAK1, transforming growth factor-β activated kinase 1; TIR, Toll/interleukin-1 receptor domain;
TLR, toll-like receptor; TRAF6, TNF receptor-associated factor 6; U, ubiquitylation. Based on [23, 24].
There are many cytokines and most of them, if not all, have pleiotropic
functions. Tumor necrosis factor (TNF), interleukin (IL)-1β, IL-6 and
CXCL8 are some of the more extensively studied cytokines, which in most
cases act as promoters of inflammation [25-27].
Apart from their effects on development, growth, and activity on other
cells, some cytokines have chemotactic abilities and are termed chemokines. One example is CXCL8, so named for the N-terminal position of
two cysteine residues separated by an undefined amino acid. CXCL8
receptors are primarily found on immune cells and the binding of CXCL8
to a high-affinity receptor initiates migration of the receptor-carrying cell
18
I MATTIAS KARLSSON towards the increased gradient of the chemokine [28]. These recruited
immune cells are mostly phagocytic neutrophils and macrophages that
engulf and destroy pathogenic microbes.
One of the non-chemotactic cytokines released from the mucosa during
infection is IL-6 [29]. Locally, IL-6 stimulates the maturation of
B-lymphocytes and increases the production of immunoglobulin A (IgA),
an important mucosal antibody class [30]. Moreover, IL-6 released during
a local infection can have numerous systemic functions. Most prominently,
it contributes to fever and initiates the release of acute-phase proteins from
the liver [31, 32]. TNF is mainly released from immune cells (macrophages,
T-lymphocytes, and natural killer cells), although it can also be secreted by
epithelial cells [33]. Studies using neutralising anti-TNF antibodies and
TNF-receptor-deficient mice have shown that TNF has a great impact on
pathogen clearance [34, 35]. Furthermore, TNF is a highly immunoactive
substance that can cause local vasodilation through the induction of nitric
oxide production [36]. This vasodilation facilitates influx of antibodies and
immune cells to the site of infection, thereby aiding in removing a pathogen. Similar to the actions of IL-6, IL-1β and TNF induce expression of
acute phase proteins and are therefore strong promoters of inflammation
[37]. IL-1β activates macrophages and takes part in the initiation of
adaptive immune responses by activating T-lymphocytes and inducing
cytokine secretion in dendritic cells [38, 39]. Moreover, pro-inflammatory
cytokines can up-regulate the expression of co-stimulatory molecules on
dendritic cells that are needed to activate T-lymphocytes [40].
Mucosal responses
Conventional immunology dictates that professional immune cells circulating in our blood or infused in tissues are responsible for the immunological
defence. Albeit immune cells are indeed crucial for a normal function of the
immune system, mucosal epithelial cells are in many cases the first cell type
that associates with pathogens and are today recognised as important in
the first steps of mounting adequate immune responses. Although most
research on mucosal immunology has been conducted on cells in the gut,
the respiratory and urogenital tract are important mucosal loci where
pathogens can enter. The structure of immunological defences within the
gut has been thoroughly described, in which lymphocytes and immune cells
are highly organised forming foci with highly specific immune cell subsets
and functions. Similar structures have been observed in the urinary tract
mucosa, with high numbers of lymphocytes and macrophages [41].
B-lymphocytes within the urinary tract produce high levels of secretory IgA
(sIgA) that can inhibit adhesion and colonisation of pathogens and are
MATTIAS KARLSSON I
19
therefore important in the maintenance of a healthy urinary tract [42]. In
addition to immune cells, epithelial cells of the urinary tract carry several
PRRs (e.g. TLR4) and produce cytokines upon contact with bacteria [33,
43]. However, although mucosal epithelial cells produce cytokines, they
secrete far less than professional immune such as macrophages found
further down in the tissue. The constantly high levels of antigens in contact
with these tissues throughout evolution are likely responsible for this
refractory state. Interestingly, epithelial cells can transport LPS from the
apical to the basolateral side, thereby feeding LPS onto the highly reactive
immune cells [44], allowing for stronger responses when such are needed.
Moreover, apart from antibodies secreted into the mucosal lumen by
B-lymphocytes and the cytokines secreted by other immune cells as well as
epithelial cells, the mucosa produces antimicrobial peptides that help to
defend the host against pathogens [45].
Responses towards microorganisms and tolerance
However marvellous our immune system seems, it is not always successful
at its job. Infections kill millions of people each day around the world, and
many more have chronic or recurrent infections. As individuals, humans
stand no chance against the favourable features of microorganisms with
their short generation times and large populations. Moreover, many people
suffer from autoimmune conditions determined by aberrations in immune
system function.
Interestingly, the host can normally tolerate a vast amount of bacteria,
for instance those that colonise our gut. Although these microorganisms
are present in close proximity to the many immune cells that make up the
mucosal-associated lymphoid tissues and express factors that on their own
would be enough to evoke an immune response (e.g. LPS) they are rarely
regarded as a threat to the host. In fact, immune signalling has proven to
be important in the development of immunological tolerance that prevents
adverse reactions towards food or other harmless substances. Mice that
lack an early adaptor protein in TLR-signalling (MyD88) are more sensitive to chemically induced (dextran sulphate sodium) damage to the gut
compared to wild type mice [46]. Moreover, mice that lack indigenous gut
microbes respond with increased mortality and morbidity when subjected
to the same chemical [47]. As it turns out, many of the proposed molecular
mechanisms of tolerance actually include regulation of NF-κB activation, a
regulation intended to prevent unmotivated pro-inflammatory responses
[48-50]. In conclusion, the same factor that largely manages responses to
pathogens is also essential when the opposite response is required.
20
I MATTIAS KARLSSON THE HUMAN MICROBIOTA
The importance of microbes
Our body is a composite of 1013 human somatic cells and 1014 microbes
and although microorganisms outnumber human cells by a factor of ten,
their total mass is limited to no more than two kilograms [51]. However,
their impact on development and continuous physiological homeostasis has
proven to be extremely significant. Apparently, these bacteria take great
part in the development of our gastrointestinal and neural system, as well
as positively affecting normal immune system development [52, 53].
Throughout our life, they continuously help with nutrient uptake, production of indispensable vitamins, and influencing immune responses. On the
genetic level, each individual carries no more than 25,000 human-derived
genes whereas the human-associated microorganisms, known collectively
as our microbiota, are estimated to contribute with three million genes
[54]. Consequently, less than 1 % of the genes present in our body are of
human origin. In light of those data, it is not at all surprising that our
microbial inhabitants so heavily influence our physiology.
Many people are frightened when confronted with the fact of being so
extensively infiltrated by microbes. Our families, friends, teachers, health
care professionals, and media have persistently told us that bacteria are
enemies and that they should be feared. And rightly so, because they can
cause severe disease and are responsible for a great number of deaths
throughout the world each day. In 1908, Paul Ehrlich and Elie Metchnikoff received the Nobel Prize in Physiology or Medicine, awarded “in
recognition of their work on immunity” [55]. The following years, Ehrlich
and his co-workers developed Salvarsan, a highly effective antimicrobial
drug used to treat syphilis. Although Metchnikoff shared Ehrlich’s interests
in bacteria, he continued in a different direction, studying the health
promoting effects of microorganisms, claiming that bacteria in yoghurt
(lactobacilli) were responsible for long life in certain sub-populations of
Eastern Europe. Though both Metchnikoff and Ehrlich contributed to the
ideas of immunology and bacteriology, Ehrlich’s beliefs prevailed and
propagated during the twentieth century: an idea that bacteria were evildoers, not needed and not wanted.
During the last decades, much more attention has been given to
Metchnikoff’s ideas and many research programmes now focus on examining the composition and function of our microbiota and health promoting
(probiotic) microbes. At the same time, the number of new antimicrobial
drugs released into the market is very limited. For long, researchers who
MATTIAS KARLSSON I
21
studied health benefits of bacteria were regarded as ostentatious and naive;
many have now demonstrated the beneficial effects of bacteria to the point
that we know that microorganisms are important for normal development.
Moreover, when things go awry, they can help maintain health and reduce
pathogenicity of disease-causing microbes.
The actual number and composition of microorganisms present in our
body as part of our microbiota has for long been debated. Culturing microorganisms by direct sampling has proven unfruitful, since the growth of
these microbes is complex and dependent on conditions that cannot be
reproduced in the lab. Metagenomic studies that do not involve microbial
culturing techniques is the latest strategy and two large-scale projects have
recently been deployed: one in the United States known as the Human
Microbiome Project (HMP) and another coordinated from the European
Union entitled Metagenomics of the Human Intestinal Tract (MetaHIT).
Both projects analyse indigenous microbes and their genes, and the
influence they have on our human somatic cells and body as a whole. So
far, such studies have collectively shown that our human microbiota
consists of multiple phyla, and that there our enormous differences in
microbial colonisation and composition between body sites and individuals
[56, 57]. Moreover, the great temporal variation of microbes adds an extra
dimension; this meta-genome can be changed, throughout the life of a
person [58]. In the future, the data harvested from these studies could be
used to expose the microbial protagonists controlling important physiological processes including disease development.
Vaginal lactobacilli and health
Most of the microbes that constitute our microbiota reside within the gut.
However, a substantial proportion of them also colonise the vagina.
Already in 1892, the German obstetrician and gynaecologist Albert Döderlein demonstrated that Gram-positive rods were present in the vagina of
pre-menopausal women. The bacterium Döderlein observed was later
named “Döderlein’s bacillus”, and it is now determined that this bacterium
is part of a complex ecosystem comprising different genera where Lactobacillus species are the most prevalent. Studies have estimated that vaginas
of pre-menopausal women carry a vast amount of lactobacilli; as much as
107 bacterial cells per millilitre of vaginal fluid is a normal finding [59].
The high levels of lactobacilli in the vagina is in sharp contrast to the
microbial composition within the lower gastrointestinal tract, where a very
small percentage (as low as 2 %) of the microbial population is made up of
lactobacilli [60].
22
I MATTIAS KARLSSON Even though lactobacilli are dominant in the vagina of most women, the
exact composition of microbes differs over time and large changes take
place throughout a woman’s life. At parturition, the vagina is sterile, but
within a few weeks, it is heavily populated by lactobacilli. These bacteria
are however only transiently present and young women demonstrate an
increased vaginal pH with low numbers of lactobacilli. It is not until the
onset of puberty, when oestrogen and glycogen levels increase, that lactobacilli again begin to dominate the vaginal microbiota. Although the
composition of the vaginal microbiota changes now and then, most women
continue to carry a Lactobacillus dominated microbiota until menopause,
when oestrogen levels and lactobacilli numbers decline. [61].
A great number of different Lactobacillus species have been isolated
from the vaginal fluid of pre-menopausal women: L. acidophilus, L. brevis,
L. casei, L. cellobiotus, L. crispatus, L. delbrueckii, L. fermentum,
L. gasseri, L. iners, L. jensenii, L. oris, L. plantarum, L. reuteri, L. rhamnosus, and L. ruminis [62-65]. Early culture-independent approaches did
however show that L. iners was the most abundant species, found in more
than 65 % of white and black women [66]. A subsequent metagenomic
study identified five vaginal community groups in Asian, white, black, and
Hispanic women [67]. Four out of the five community groups were rich in
lactobacilli: L. crispatus, L. gasseri, L. iners, and L. jensenii. The fifth
group was phylogenetically diverse, and although it included lactobacilli
for most women, other genera such as Atopobium and Streptococcus
dominated. This diverse group was especially prevalent in black and
Hispanic women, two groups that more frequently than others displayed
high vaginal pH and clinically established bacterial vaginosis (BV). Many
women suffer from BV, a usually painless condition characterised by malodorous vaginal discharges associated with an elevated risk for pre-term
birth and increased susceptibility to HIV and other sexually transmitted
infections [68]. Other studies have shown that episodes of a microbial
composition low in lactobacilli are linked to urogenital disease such as BV
and an increased susceptibility to overgrowth of opportunistic pathogens
that normally only constitute a minute proportion of the microbiota [69].
Many of the protective actions of vaginal lactobacilli have been
attributed to the low pH (less than 4.5), which is maintained by lactic acid
production in lactobacilli through the fermentation of sugars [70]. Even
though lactobacilli thrive in an acidic environment, the growth of urogenital pathogens such as E. coli is inhibited by a low pH [71, 72]. Apart
from the low pH as such, lactic acid has been shown to directly target
Gram-negative bacteria such as E. coli by disrupting the outer membrane,
facilitating the entry of antibacterial compounds [73]. Moreover, more
MATTIAS KARLSSON I
23
than half of vaginally isolated lactobacilli produce hydrogen peroxide, a
toxic product that adversely affects urogenital pathogens including
Gardnerella vaginalis, a microbe highly associated with BV [65].
Another important feature of autochthonous microorganisms is their
ability to competitively exclude other microbes. Human-associated
microbes colonising the mucosa, such as lactobacilli, efficiently adhere to
the mucosal epithelial cells [74] and use up nutrients during growth. By
doing so, indigenous microbes can simply competitively exclude the adhesion and growth of potential pathogens. Since efficient proliferation and
adhesion to host cells is a strong prerequisite of causing an infection, high
levels of non-pathogenic lactobacilli are therefore able to prevent disease.
24
I MATTIAS KARLSSON PROBIOTICS
According to one definition, as proposed by the World Health Organization (WHO) and the Food and Agriculture Organization of the United
States (FAO), probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit on the host” [75]. The
definition is accompanied by a set of guidelines, requirements that need to
be met, in order to classify a product as probiotic. According to these
guidelines, the candidate microorganism has to be identified down to strain
level and its safety and efficacy established using double-blind, randomised,
placebo-controlled studies. Furthermore, the probiotic has to be compared
to conventional treatment of the clinical condition for which the probiotic
is aimed. Finally, the consumers need to be informed about the health
benefits when they buy a product. Decreed health claims should be clear
and the number of microorganisms that are needed in order for a desirable
effect (dosage) should be written on the product along with shelf life,
storage conditions, and contact information.
Within the European Union, a new regulation (Regulation (EC) No
1924/2006 of the European Parliament and of the Council) provides guidelines on food health claims. The European Food Safety Authority (EFSA)
has since the adoption of this regulation had to even-handedly evaluate
numerous health claims from companies adding microorganisms to food
components. In short, consumers have to gain from choosing the probiotic
product as opposed to a non-probiotic alternative. In addition to just being
nutritious, the health effect of the product has to come from the live
bacteria. It is believed that by using this stringent and coordinated
approach from authorities, consumers will be given a more complete
description of the probiotic along with validated health claims similar to
those of pharmaceuticals.
The Russian Nobel laureate Elie Metchnikoff was one of the first to
acknowledge bacteria as important in sustaining human health and studies
on longevity in the early nineteen hundreds had convinced him that
bacteria play a crucial role in quality of life and protection from pathogenesis. He did however not coin the term probiotic. The earliest finding of the
term is according to Hamilton-Miller et al. [76], in a publication from
1953 by Werner Kollath who described “probiotika” as organic and inorganic substances that could restore health. The following year, Ferdinand
Vergin proposed that fermentation products could be used as probiotics,
which was the first step of associating microorganisms to probiotics. In
1965, Lilly and Stillwell suggested another meaning of probiotics: “substances secreted by one microorganism which stimulates the growth of
MATTIAS KARLSSON I
25
another” [77]. Nearly ten years later, in 1974, Parker defined probiotics as
“organisms and substances which contribute to intestinal microbial
balance” [78]. Thus, during a time span of 20 years, the term probiotic had
developed from a general description of a supplement important for
microbial growth or general health to a precise description of characterised
microorganisms able to improve specific aspects of health. With increased
research on probiotic function, the current definition by WHO/FAO will
most likely be revised in the near future as well.
One of the earliest examples of a probiotic in agreement with the latest
definition is the E. coli strain Nissle 1917, named after the German
physician Alfred Nissle. Nissle demonstrated a profound interest in the
antagonistic effects of E. coli on pathogenic microbes and in his work, he
isolated numerous E. coli strains with varying effects on intestinal pathogens. In 1917, he acquired faecal samples from soldiers fighting in World
War I that remained healthy during outbreaks of intestinal diseases. Nissle
enclosed the bacteria in gelatine capsules and it was subsequently launched
as the product Mutaflor. It is now one of the most studied probiotic
preparations and has been used to treat a number of different intestinal
disorders. But how can one bacterium bring about so many positive
changes? A study on the effects of E. coli Nissle 1917 on enterocytes
showed that approximately 300 genes were modulated by the bacterium,
many of them involved in host response including immune activation [79].
Most probiotics today, however, belong to the genus of Lactobacillus.
There are many health claims for products containing these microorganisms, most of them related to gut function – as products supporting
bowel regularity and prevent diarrhoea or to treat diseases such as
ulcerative colitis, Crohn’s disease, and irritable bowel syndrome. Clinical
data are compelling; many probiotics can positively affect gut function. As
in the case with E. coli Nissle 1917, a large proportion of the effects
following Lactobacillus probiotic therapy have been attributed to changes
in immune function as well as the production of antimicrobial substances
that can inhibit growth of other microorganisms [80].
Immunomodulation by probiotics
The microbiota has a fundamental role in immune system development. By
colonising our body early in life, these microorganisms aid our immune
system in properly dealing with environmental cues, distinguishing between
pathogenic and commensal/mutualistic organisms. Since a majority of probiotic microorganisms are derived from our own microbiota it is to be
expected that they too have immunomodulatory abilities. Accordingly,
26
I MATTIAS KARLSSON studies have shown that probiotic microbes affect immune responses, both
innate and adaptive.
On the cellular level, probiotics can induce diametrically opposite outcomes within the host. Lactobacilli can promote TNF release from mouse
monocytes to a varying degree mainly due to lipoteichoic acid (LTA)
present in the Gram-positive cell wall [81]. These polysaccharides are
frequently decorated with D-alanine, which contribute significantly to the
overall impact of LTA on the bacterial cell in terms of structure and
survival [82] as well as its immunomodulatory abilities (i.e. the ability to
activate TLR2 pathways). Genetically modified Lactobacillus plantarum
that do not incorporate D-alanine into its LTA are poor activators of TNF
release in human immune cells compared to a wild-type strain with Dalanine-rich LTA [83].
Although heat-killed lactobacilli preparations readily induce an inflammatory response on the cellular level, studies on viable bacteria have
shown that they can effectively down-regulate immune responses through
various pathways including NF-κB [50], most likely due to other
substances that are produced and released by the bacteria during their
growth. Unfortunately, very few of these studies have further characterised
the immunomodulatory substances. In fact, lactobacilli release a very
limited number of proteins during growth, and only a few of them have
success-fully been associated with changes in immune activity.
L. rhamnosus GG (one of the most studied Lactobacillus strains) secretes
two proteins, p40 and p75, which successfully prevent TNF-induced
damage and apoptosis in human and mouse colon cells [84]. Moreover,
these proteins can protect intestinal epithelial cells from hydrogen peroxide-induced damage in a process involving MAP kinases, although the
role of NF-κB is still unclear [85]. Elongation factor Thermo unstable (EFTu) and chaperonin GroEL, two extracellular products isolated from
L. johnsonii have also been described to induce pro-inflammatory CXCL8
release from human cells [86, 87].
Apart from the local cellular responses, lactobacilli can both up- and
down-regulate systemic immune responses [88]. L. casei has previously
been reported to influence B-lymphocyte functions and can elevate antibody production in mice infected with Salmonella or E. coli, thereby
protecting them from disease [89]. Furthermore, short immunostimulatory
DNA sequences from L. rhamnosus GG are able to skew T-lymphocyte
responses and decrease IgE production in mice by acting through TLR9
[90]. Moreover, lactobacilli can greatly influence the physiology of
dendritic cells (key players in instigating adaptive responses), with both
pro- and anti-inflammatory outcomes [91].
MATTIAS KARLSSON I
27
Additional probiotic effects
In addition to immunomodulation, many studies have explored the direct
interplay between probiotic microorganisms and pathogens. Competition
for nutrients and space at the mucosa is a well-known beneficial outcome
of commensal colonisation. It should be noted that although competitive
exclusion is thought to be a major determinant in preventing pathogen
entry, probiotics are usually poor colonisers and although they can effectively adhere to tissue cells and compete for nutrients, they are in most
cases cleared from the host within weeks [92].
Apart from competitive exclusion, antimicrobial substances produced by
probiotics can negatively affect the growth of several human pathogens.
The most studied group of these antimicrobial agents are the bacteriocins,
narrow-spectrum antibiotics produced by many microbes, typically targeting species phylogenetically close to the microorganism in which the
bacteriocin was produced. However, bacteriocins from a vaginally isolated
L. salivarius strain can kill both Gram-positive Enterococcus species and
Gram-negative Neisseria gonorrhoeae through pore formation and lysis of
bacterial cells [93]. The probiotic effect of another L. salivarius strain has
been found to exclusively rely on the production of a bacteriocin [94].
While mice given bacteriocin-producing L. salivarius were protected from
Listeria monocytogenes infection, the bacteriocin null mutant offered no
protection. Moreover, probiotics can disrupt protective biofilm structures
of Gardnerella vaginalis [95]. The biofilms challenged with L. crispatus, L.
iners and a probiotic L. reuteri strain were significantly reduced. This biofilm-disruptive process was not dependent on lactic acid production (low
pH) or hydrogen peroxide, although both pH and lactic acid are known to
inhibit the growth of certain microbes.
Safety issues
Consumption of probiotic products is rarely linked to infection and many
strains are generally regarded as safe when consumed orally. Moreover,
similar microorganisms are frequently used in food production such as
dairy products including yoghurt and cheese. Even though these microbes
have an exceptional safety record, there are concerns that lactobacilli and
other microorganisms used as probiotics or in food may involve health
risks.
The EU-project “Biosafety Evaluation of Probiotic Lactic Acid Bacteria
used for Human Consumption” (PROSAFE), has studied safety issues related to probiotics and suggested several areas that need to be taken into
account when discussing their use. Although correct identification and
28
I MATTIAS KARLSSON characterisation of the probiotic is essential as a natural first step in probiotic safety, the virulence of probiotic microorganisms and the possibility
of them carrying antibiotic resistance has emerged as two important
concerns [96].
The virulence of available probiotics is generally considered very low
and luckily there are a very limited number of reported cases of infection in
individuals consuming probiotics. Nevertheless, a few events of sepsis
caused by probiotic L. rhamnosus GG therapy in children and adults have
been described [97]. The general outcomes have however been mild with
full recovery and many patients enrolled in probiotic studies have at the
time of trial been predisposed, suffering from risk factors such as diabetes,
cancer, and prematurity. A recent drawback in probiotic implementation
came with a clinical nation-wide study (PROPATRIA) in the Netherlands
assessing the potential use of six different Lactobacillus, Lactococcus, and
Bifidobacterium strains (L. acidophilus, L. casei, L. salivarius, Lactococcus
lactis, Bifidobacterium bifidum, and B. infantis) in pancreatitis treatment
[98]. The strains were selected based on their in vitro abilities to inhibit the
growth of pathogens associated with pancreatitis and were regarded as
safe. The study outcome was 24 deaths in the probiotic group compared to
9 deaths in control subjects not receiving the study product. Mesenteric
ischemia within the probiotic group was a major finding during autopsy as
well as inflammation in the small bowel. This study clearly underlines the
benefits of rigorous characterisation of the strains to be used in a probiotic
formulation, not only their effect on other bacteria (which was an
important selection criterion for the strains used in the study), but also
their impact on host cells and immunity. It should be noted that the bacteria in this study were not consumed orally as probiotics normally are administered, and that they were used in critically ill patients, a known risk
factor for complications following probiotic use.
Intrinsic antimicrobial resistance in lactobacilli and other lactic acid bacteria has been of some concern, especially the potential of transferring
resistance genes to pathogenic bacteria [99]. Initial laboratory findings
have shown that lactobacilli can potently transfer tetracycline resistance
genes to other Gram-positive bacteria [100]. However, a large survey of
anti-microbial susceptibility in gut microbes, emanating from the PROSAFE-project demonstrated that most autochthonous lactobacilli are
susceptible to antibiotics, with very few exceptions [101]. Moreover,
resistance transfer to other genera within a host seems to be limited and
tetracycline resistance from L. reuteri to gut microbes in the gastrointestinal passage could not be detected in a recent study [102].
MATTIAS KARLSSON I
29
Altogether, considering the extensive number of probiotic products sold
each day and their possible health benefits, the net gain of probiotic use
remains high. With the very few reports on adverse effects including virulence and antibiotic resistance, probiotics can in general be determined as
safe for consumption in otherwise healthy individuals.
30
I MATTIAS KARLSSON URINARY TRACT INFECTIONS
UTI is one of the most common bacterial diseases, affecting billions of
women each year worldwide. Based on case records from the United States,
it is estimated that approximately 400,000 women in Sweden suffer from a
UTI each year and are responsible for 1 % of primary health care visits
[103]. The symptoms during a UTI may vary from urgency to pain during
micturition although a great proportion of women with UTI are asymptomatic. The severities of UTI symptoms are typically dependent on the site
of infection. While infections in the lower urinary tract affecting the
bladder (cystitis) correlates with milder symptoms, ascension to the kidneys
(pyelonephritis) is a potentially fatal condition where bacteria are released
into the blood stream (sepsis). Apart from the individual suffering, the
costs for society are considerable. Statistically, reproductive age women
have a 50 % risk of developing a UTI during their lifetime [104]. For those
who have suffered one UTI, the risk of recurrence is substantial and can
occur as frequently as several times per year [105]. Moreover, although
treatment options such as antibiotics are usually effective, recurrent infections are hard to control and long-term treatment is associated with side
effects.
UTI pathogenesis
The main risk factors for UTI recurrences are related to sexual activity;
both frequency of intercourse and new sex partners are strong risk factors
contributing to UTI development, likely due to deposition of uropathogens
into the urogenital area during sex [106]. Although UTIs are dependent on
colonisation by microbes, there seems to be a strong genetic component
governing the overall risk of suffering from a UTI. A history of UTI in the
mother is associated with increased risk for recurrent UTIs, and patients
suffering from recurrent UTIs have lower TLR4 expression on monocytes
than healthy controls suggesting they have a reduced ability to sense pathogens [107]. Furthermore, mice with non-functional TLR4 are more
susceptible to infections with uropathogens [108]. Certain polymorphisms
in CXCR1, a high-affinity receptor for CXCL8 have also been linked to
asymptomatic bacteriuria [109]. Thus, albeit environmental factors (uropathogenic microbes) are essential for UTI development, the genetic background can obviously influence an individual’s susceptibility to colonisation.
Infections with pathogenic microbes are followed by inflammation, a
host’s response in which the body defends itself and try to eradicate the
pathogen. Many pathogens try to evade this response, thereby circum-
MATTIAS KARLSSON I
31
venting detection and an inflammatory reaction. Although infections with
uropathogenic E. coli (UPEC) are associated with inflammation and
disease symptoms, they are far better at subverting the host reaction
compared to non-virulent strains, and can effectively inhibit cytokine
release from urinary bladder epithelial (urothelial) cells [110]. Some UPEC
can effectively be internalised into those tissue cells, possibly by several
different mechanisms involving both fimbriae and toxins including cytotoxic necrotising factor 1 [111]. By hiding within cells, bacteria are
protected from circulating immune cells and antibodies, and can migrate
out of the cell and initiate a new infection when the conditions are right.
Other ways of evading the immune system is by direct destruction of
immune cells. Some UPEC carry a specific virulence factor known as
α-hemolysin, which preferentially kills immune cells thus hampering the
host’s ability to fight off pathogens [112].
Recently, UPEC-specific proteins mimicking the intracellular part of the
TLR4 protein known as the toll-interleukin 1 (TIR) domain have been
isolated [113]. These TIR-containing proteins (TCPs) are secreted by the
bacteria and subsequently bind to the intracellular part of TLR4, thereby
blocking the further signalling pathway required for induction of
inflammatory genes caused by LPS or other TLR4-binding structures.
Given the importance of TLR4 in UTI pathogenesis, such results are
extremely interesting.
P pili are adhesive molecules found on some UPEC, especially those
causing pyelonephritis (hence the name P pilus). These pili readily attach to
cells found in the kidneys facilitating adhesion of the microbe. Moreover,
such pili can hamper the migration of secretory IgA across the mucosal
epithelium into the mucosal lumen, normally found in high concentration
in the gastrointestinal and urogenital tract [114]. Reduced transmigration
of secretory antibodies through the epithelial barrier is thought to impair
activation of immunity in the urogenital tract and adversely affect disease
outcome. The immune suppressive features of UPEC are summarised in
Figure 2.
32
I MATTIAS KARLSSON Figure 2. Summary of UPEC immune suppressive features.
Approximately 80 % of all UTIs are caused by UPEC, 5-15 % by Staphylococcus saprophyticus, while a few patients have growth of Proteus or
Klebsiella species [115]. These are mainly thought to emanate from the
host microbiota or a sexual partner. Even though lactobacilli inhabit the
urogenital mucosa, they are rarely the cause of UTIs. One case report
identified an L. delbrueckii strain as the cause of UTI in an 85-year-old,
otherwise immunocompetent woman and another case study described a
66-year-old man suffering from septic UTI caused by L. gasseri [116, 117]
concluding that UTIs caused by lactobacilli occur but are very rare and
their colonisation is usually not associated with disease. Although there
was no further genetic characterisation of these UTI-causing strains, such
approaches could be used to expose underlying genetic differences between
pathogenic versus non-pathogenic strains of bacteria.
MATTIAS KARLSSON I
33
Urogenital probiotics
Most probiotics on the market are used to treat or prevent gastrointestinal
disorders while only a limited number of bacteria are used as extraintestinal regimens. Although there are very few specific products on the
marked suggested to treat or prevent UTIs, a study comparing dietary
factors influence on UTI recurrences found that consumption of fermented
milk products such as yoghurt significantly reduced episodes of UTI [118].
There are several putative mechanisms by which lactobacilli could help
to prevent or treat infections of the urinary tract. Most UTIs are caused by
faecal contaminants entering the urinary tract travelling pass the perineum,
and females are at extra risk due to the close proximity of the urethral
orifice and anus, as well as the short length of their urethra. Furthermore,
faecal contaminants are readily introduced during sexual intercourse. The
microbiota within the intestine is thus of great importance. By changing the
gut microbial composition into one that lacks UTI-causing E. coli, the
overall risk of an UTI would decrease. Unfortunately, there has been little
attention given to the role of the gut microbiota and UTI risk. Although
many uropathogens are thought to be derived from the woman’s gut
microbiota, there have been no large studies exploring the eventual
differences in gut microbial composition between women that are predisposed for developing UTIs compared to women who remain free of such
infections. Consequently, it is currently unknown if probiotics could be
used to alter gut microbiotas into ones with fewer bacteria expressing UTI
virulence factors.
Moreover, orally consumed probiotics that survive the gastrointestinal
passage can end up in the vagina and from there directly exert their effects,
on both pathogenic microbes and the host. Interestingly, women with a
history of recurrent UTIs have significantly more vaginal colonisation of
E. coli compared to healthy subjects. There is an inverse relationship of
hydrogen peroxide-producing lactobacilli and the E. coli vaginal colonisation proving the importance of lactobacilli in UTI pathogenesis [119].
Moreover, vaginal administration of oestrogen, which supports the growth
of lactobacilli, can reduce UTI recurrence in postmenopausal women [120].
The observant critic explicitly demarcates vaginal lactobacilli from lactobacilli present in the urinary system, e.g. the urethra and bladder. Although
vaginal lactobacilli have been thoroughly examined, the colonisation of the
urinary system is still not fully understood. Lactobacilli have however been
cultured from the female urethra [121] and metagenomics have
subsequently found high levels of lactobacilli in both the urethra and urine
of healthy male subjects [122, 123].
34
I MATTIAS KARLSSON One of the few probiotic formulations used for urogenital diseases is a
combination of two Lactobacillus strains, L. rhamnosus GR-1 and
L. reuteri RC-14 marketed by Chr. Hansen (Denmark) under the trade
name Urex and Jarrow Formulas as Fem-dophilus (United States). This
bacterial combination has been evaluated for a number of urogenital conditions including BV, yeast infection, and UTI [80]. Both L. rhamnosus
GR-1 and L. reuteri RC-14 transiently colonise the vagina when they are
administered orally or vaginally [124, 125]. Efficient colonisation is
believed to be an important probiotic trait, if the effects rely on direct displacement of pathogens or actions on the urinary tract mucosa (e.g. influencing immune responses). Initial studies on L. rhamnosus GR-1 showed
that it could prevent UPEC colonisation in rat bladder and thereby prevent
the onset of UTI [126]. Moreover, in a small trial of 41 women being
treated with antibiotics for UTI, vaginal administration of L. rhamnosus
GR-1 and L. reuteri B-54 reduced UTI recurrence after antibiotic therapy
with 50 %, showing that urogenital probiotics can be used therapeutically
[127]. In another study, the same combination of microbes was given intravaginally to women suffering from recurrent UTIs for twelve months,
which resulted in a reduced number of UTI recurrences [128]. From having
approximately six UTI episodes the year before enrolment, their UTI
episodes were reduced to 1.6 the year after probiotic therapy.
The probiotic L. crispatus CTV-05, marketed as LACTIN-V by Osel
(United States), is a vaginally derived Lactobacillus strain that has recently
shown great potential to reduce recurrence of UTI in women. Taken as a
vaginal suppository, it is rarely associated with adverse effects apart from a
few patients exhibiting a low-grade inflammation [129]. In a phase two
study, colonisation of L. crispatus CTV-05 in the vagina was associated
with decreased UTI recurrence [130]. Within the study group given the
probiotic, only 15 % of women suffered from another UTI during the
course of the study, compared to 27 % in the placebo group. Another
L. crispatus strain (GAI 98332) was in a pilot study evaluated for its
impact on UTI recurrence [131]. Women experiencing frequent UTIs were
instructed to vaginally insert a suppository containing L. crispatus GAI
98332 every two days for one year. The women had a mean value of five
UTI episodes per year before probiotic treatment, a number that was
reduced to 1.3 after one year of Lactobacillus instillation.
Since most UTIs are cleared within a short period of time, they do not
need extensive treatment. In Sweden, the primary pharmaceutical treatment options are penicillin (pivmecillinam) and nitrofurantoin, although
other antibiotics are frequently used in practice [132]. A substantial
proportion of uropathogenic isolates carry antibiotic resistance and it is
MATTIAS KARLSSON I
35
therefore important to develop new treatment strategies. Interestingly, a
study of children with frequent UTI episodes found that probiotics prevented new infections just as efficiently as antibiotics, showing there are
alternatives to antibiotic therapy [133].
While some lactobacilli, such as L. rhamnosus GR-1 and L. crispatus
CTV-05 have been associated with UTI protection, a study comparing
cranberry juice with orally administered L. rhamnosus GG, showed that
this intestinal probiotic strain was unable to reduce UTI recurrence [134].
Since L. rhamnosus GG does not even transiently colonise the vagina when
given vaginally [124], these results might simply reflect shortcomings in
vaginal colonisation highlighting the importance of such features.
Although few clinical trials have been performed to date, there is ample
evidence motivating further studies on probiotics as a means of reducing
UTI occurrence. Moreover, it has been pointed out that already conducted
trials have been underpowered and therefore unable to reach statistically
significant results although the results per se are interesting and support the
use of probiotics. The Cochrane Collaboration has recently instigated a
meta-analysis on the use of probiotics in prevention of UTI in children and
adults, although it is not yet completed [135]. While antibiotics are usually
beneficial in the acute disease, increased antibiotic resistance and the side
effects of persistent antibiotic use in patients suffering from recurrent
infections require new treatment strategies.
36
I MATTIAS KARLSSON METHODOLOGY
Cell challenges
Culture of cells and bacteria
All data throughout this thesis is based on the culturing and challenging of
human eukaryotic cells (urinary bladder epithelial cells and macrophagelike cells) in vitro. The use of cell lines in basic biological research is
invaluable, since it gives consistency of data and through the international
distribution by culture collections such as the American Type Culture
Collection (ATCC), acquiring cell lines is easy. These cells have been
selected because of their ability to be easily cultured in a medium with a
surplus of nutrients at the right temperature and pH. Two urothelial cell
lines were used in the five studies: T24 (ATCC HTB-4) and 5637 (ATCC
HTB-9), both derived from the transitional epithelium of human bladders.
They respond to E. coli stimulus and are for that reason frequently used in
the study of the cellular pathogenesis during UTI. The immune cells in
study IV were human monocytic cells isolated from a leukemic patient,
now distributed under the name THP-1 (ATCC TIB-202). These cells
normally grow in suspension, although they can be differentiated into nondividing, adhering cells [136]. By the addition of phorbol 12-myristate 13acetate (PMA), they differentiate into adherent cells with increased
expression of macrophage-specific markers, phagocytosing abilities, and
loss of proliferation.
The bacteria used in the studies were mainly lactobacilli, predominantly
L. rhamnosus GR-1 (ATCC 55826) and GG (ATCC 53103), as well as
L. reuteri RC-14 (ATCC 55845). These are patented probiotic bacteria,
intended for various ailments. Lactobacilli used in Study V are type strains
for various species within the genus, except for L. acidophilus NCFM
(ATCC 700396), which is also a patented probiotic. Lactobacilli require
complex nutrient conditions for successful growth, including reduced levels
of oxygen. The nutrient requirements were met by using a specific culture
medium with added growth factors sustaining Lactobacillus growth. The
E. coli GR12 strain is a pyelonephritis isolate expressing adhesins such as
P pili, and is readily grown in nutrient broth [137]. While several of the
studies in in this thesis use viable lactobacilli, E. coli were always heatkilled before added to the cells. The primarily use of heat-killed E. coli was
to promote an immunological response from the eukaryotic cells viz.
NF-κB and cytokine expression. Heat-killed bacteria delivered LPS and
other heat-stable components to the eukaryotic cells, with profound effects
MATTIAS KARLSSON I
37
on cellular immunological reactions mimicking those during an in vivo
infection. Although viable E. coli can be used to challenge cells, live
bacteria introduce yet another level of complexity to the experiments
through eventual manipulation of E. coli physiology by lactobacilli during
co-culture.
Isolation and fractionation of released products from lactobacilli
Most of the work in this thesis is based on the use of viable lactobacilli and
heat-killed E. coli. However, in order to learn more about the factors
controlling cellular innate immune responses, Lactobacillus material
released from the bacteria during growth was isolated in Study I, II, and
III. The isolation of released products from lactobacilli was performed
using a large number of washed viable bacteria (1013) transferred to a
phosphate buffered saline (PBS) solution. The bacteria were allowed to
release products into this saline solution for a short period of time, before
removal of the bacterial cells through centrifugation and filtration. The use
of released products eliminated specific actions depending on cell wallassociated products from bacteria that would otherwise be present during
challenge of tissue cells. Moreover, the use of released products excluded
actions based solely on pH variation in the tissue culture medium due to
Lactobacillus fermentation. The resultant solution contained products that
varied a lot in size. To separate the products into fractions based on their
native size, centrifugal concentrators with semi-permeable membranes of
different pore size were used. The approach created five separate fractions
and each fraction contained native proteins within a specific size range.
These fractions were subsequently added to the cell culture, in order to
evaluate their biological activities.
Co-culture of urothelial cells and macrophage-like cells
In studies I, II, III, and V, adherent cells were grown in monoculture, using
only one cell type at a time. The cells were cultured on a surface where
they eventually produced a monolayer of cells, onto which any agent that
needed to be tested was added. There is a complex interplay between
different cell types in normal physiological responses. During infection of
the urinary bladder with pathogenic microbes, epithelial cells release
chemoattractant molecules (e.g. chemokines) to which immune cells
respond. To characterise responses from urothelial cells and macrophagelike cells during challenge with bacteria, cells were grown in two “wells”,
separated by a semi-permeable membrane of polyethersulfone with pores
of 0.2 µm (Figure 3). This allowed small molecules to diffuse between the
two cell types, although they were physically separated. Urothelial cells
38
I MATTIAS KARLSSON (5637 cell line) were grown on the top membrane, while macrophage-like
cells were cultured on the bottom well. Although several bladder models
have been fashioned, with very intricate three-dimensional structures, none
of them have incorporated immune cells. The relatively simple model in
Study IV, allowed scoring of effects on both urothelial cells as well as
immune cells.
Figure 3. Representation of co-culture setting with 5637 urothelial cells and THP-1
macrophage-like cells in Study IV.
Detection of immunological outcomes
While the challenging of cells was rather straightforward (by adding either
Lactobacillus cells or material in the absence or presence of heat-killed
E. coli), the immunological outcomes were assayed through a variety of
methods. The specific methods used are schematically summarised in
Figure 4.
MATTIAS KARLSSON I
39
Figure 4. Overview of the experimental setup and different methods used to
examine the immunomodulatory actions of lactobacilli. 1 Released products from
lactobacilli as well as live or heat-killed bacteria were used to stimulate cells.
2
ELISA was used to study released cytokines and cytokine receptors.
NF-κB activation (luciferase assay)
NF-κB dimers are transcription factors that depend on translocation into
the nucleus where they facilitate the expression of different genes by binding to specific sequences on DNA. The dimers are however continuously
produced and transported out of the nucleus into the cytosol, where they
are bound to inhibitor proteins that upon the appropriate molecular cues
(such as TLR activation) are detached from the NF-κB dimers. The NF-κB
transcription factor carries a specific signal directing it to the nucleus once
the protein complex is no longer bound to inhibitors. To determine how
much NF-κB has been translocated one can measure how efficiently it
binds and activates specific NF-κB binding sequences within the cell
nucleus. Throughout this thesis, when NF-κB translocation has been
determined, it has been based on data from synthetic NF-κB binding
sequences that are introduced into the cells by means of transfection.
Briefly, a vector with NF-κB binding sequences coupled to a luciferase gene
from fireflies was introduced into the cells in multiple copies. To facilitate
uptake, the circular vectors were coated into lipid spheres, which protect
40
I MATTIAS KARLSSON the DNA and subsequently transport them into the cells. A small proportion of these vectors end up inside the nucleus of the target cell. When
endogenous NF-κB dimers are activated and translocated into the nucleus,
they will attach themselves to the NF-κB binding sequences on the vector,
leading to the production of luciferase. Luciferase is an enzyme that catalyses a reaction of luciferin conversion into oxyluciferin and light. By
measuring light production after luciferin addition using a highly sensitive
luminometer, one can determine how much free NF-κB has been translocated. Furthermore, to control for differences in transfection efficiency, a
second vector with other binding sequences, that continuously produce
another luciferase molecule from Renilla reniformis, was concomitantly
transfected into the cells. Luckily, the two luciferase enzymes have different
requirements for their function and can therefore be independently
assayed. A normalised value of the luciferase activity from firefly and
Renilla luciferase was obtained by simply calculating a ratio of the two,
and expressed as relative activation of the transcription factor.
Quantitative PCR
The central dogma of molecular biology dictates that the genetic flow of
information is from DNA to RNA, followed by protein synthesis.
Messenger (m)RNA precedes protein synthesis and quantifying the amount
of mRNA present within cells is routinely used to assay the effects on gene
transcription. In Study II and IV, mRNA was isolated from Lactobacillus
and E. coli-challenged cells using silica, which efficiently binds RNA under
specific conditions. Since mRNA is highly unstable, it was first converted
to more stable complementary DNA through reverse transcription.
Through the use of specific primers and a fluorescent dye (SYBR Green)
that exclusively binds to double-stranded DNA formed during PCR, a
photomultiplier tube can detect incorporation of SYBR Green and thus the
increased number of copies. The method allows several conditions to be
run in parallel, thereby allowing detection of several treatment conditions
and genes near-simultaneously. To adjust for variation of initial gene
copies between replicates, all genes of interest were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels.
Fluorescence microscopy and native immunoblots
In order to analyse the levels of TLR4, monoclonal antibodies conjugated
to the fluorophore fluorescein isothiocyanate (FITC) or horseradish peroxidase (HRP) were used. FITC-labelled antibodies were used to find surface
TLR4 expressed on T24 urothelial cells grown on a cover slip. To better
visualise the cells in microscopy, a dye that specifically binds to DNA
MATTIAS KARLSSON I
41
(DAPI) was added to fixed cells. Moreover, phalloidin coupled to another
fluorophore (AlexaFluor555) that emits orange light (maximum emission
at 565 nm) upon excitation was used to detect actin filaments within the
cells. HRP-conjugated antibodies were used to semi-quantitatively determine the presence of TLR4 protein in spots of lysed T24 cells from the
different treatments, visualised through chemiluminescence by oxidation of
luminol.
ELISA
Most of the results presented in this thesis rely on the concentration of
cytokines secreted from eukaryotic cells. The concentration of secreted
proteins in cell culture supernatants was quantified using commercially
available enzyme-linked immunosorbent assays (ELISAs). All assays were
based on a sensitive and reliable sandwich method, where two separate
antibody molecules are used. While the primary antibody is used to capture
the protein of interest, the secondary antibody is directly or indirectly
coupled to an enzyme, in this case HRP. Unbound protein and detection
antibody was removed through washing before the amount of protein was
quantified by addition of a chromogenic substrate and absorbance readings. Absolute concentrations were obtained by comparing the absorbance
readings to a standard curve with known concentrations of recombinant
protein.
42
I MATTIAS KARLSSON RESULTS AND DISCUSSION
The results presented and discussed in this section are presented in subheadings that make up key findings from the five studies (Study I-V) on
which this thesis is based. The results are presented and discussed in subheadings that make up key findings from the individual studies. Study I
described NF-κB reactions in urothelial cells following L. rhamnosus GR-1
and L. reuteri RC-14 treatment. Study II focused on TNF and NF-κB
regulation by L. rhamnosus GR-1 from a mechanistic perspective. Study III
further characterised the immunomodulatory factors present in the released
products from L. rhamnosus GR-1. In Study IV, the cytokine-regulating
effects of L. rhamnosus GR-1, on both urothelial cells and macrophagelike cells were analysed. As L. rhamnosus GR-1 was able to modulate
innate responses from urothelial cells, it was of interest to determine the
effects of other species within the genus. Therefore, in Study V, a number
of Lactobacillus species were evaluated based on their abilities to change
the levels of CXCL8, a chemokine of great importance in UTI pathogenesis. Figure 5 summarises the different studies in terms of study objects,
challenging conditions, and endpoints.
Figure 5. Overview of the five studies, with information on the bacteria used, their
eventual preparation, the measured outcome, and the stimulated cell type (* Heatkilled bacteria and isolated cell wall material, 1 TNF, IL-6, and CXCL8; 2 TNF, IL1β, IL-6, and CXCL8).
MATTIAS KARLSSON I
43
The urogenital probiotic L. rhamnosus GR-1 influences tissue cell
responses to E. coli
During the normal course of a UTI, the cells of the urethra and bladder
respond to pathogenic bacteria (of which most are uropathogenic E. coli)
by releasing substantial amounts of cytokines that promote an
inflammation. Many researchers have studied the effects of lactobacilli on
cytokine secretion, both in vitro and in vivo, although few have been
interested in the direct effects on the mucosal epithelial cells from the
urinary tract.
In study I, the impact of probiotic L. rhamnosus GR-1 and L. reuteri
RC-14 on NF-κB activation in urothelial cells was evaluated. The urothelial cells (T24 cell line) used in Study I had significant activation of the
NF-κB pathway after E. coli challenge. Released products from
L. rhamnosus GR-1 and L. reuteri RC-14 isolated in PBS were added to
the urothelial cells, either resting or challenged with heat-killed E. coli.
While L. reuteri RC-14 products did not alter the activation of NF-κB,
released products from L. rhamnosus GR-1 significantly augmented NF-κB
activation in E. coli-challenged cells (Figure 6).
Figure 6. NF-κB activation in non-stimulated and E. coli-stimulated urothelial cells
subsequently challenged with released products from L. rhamnosus GR-1 or
L. reuteri RC-14. P-values <0.05 compared to (a) non-stimulated or (b) E. colistimulated cells. Reproduced from [138].
44
I MATTIAS KARLSSON Furthermore, while this augmentation of NF-κB activation was present
when using live bacteria, heat-killed lactobacilli only gave a very small
induction of NF-κB, as was shown in Study II (Figure 7).
Figure 7. NF-κB activation in E. coli-challenged urothelial cells concomitantly
stimulated with viable (V) or heat-killed (HK) L. rhamnosus GR-1. P-values <0.05
compared to (a) un-stimulated cells or (b) E. coli-challenged cells. Reproduced from
[139].
L. rhamnosus GG has previously been shown to induce NF-κB activation
in human macrophages by Miettinen et al. [140] while other studies have
demonstrated the opposite effect in intestinal epithelial cells [141]. Moreover, van Baarlen et al. have shown that L. rhamnosus GG preferentially
affects MAP kinase pathways in the human small intestine while NF-κB
pathways are not that readily induced [142]. It is possible that the effects
on NF-κB in Study I and II reflect some fundamental differences between
intestinal epithelial and urothelial cells. Furthermore, although
L. rhamnosus GG was able to augment NF-κB in Study II, it was not as
potent as L. rhamnosus GR-1, reflecting differences between the two
strains.
NF-κB is important in the initial immunological responses following
pathogen contact in almost all cell types, including epithelial cells of the
urinary tract. As the first line of defence, it is imperative that they respond
by producing a battery of cytokines, some of them regulated by NF-κB. As
selected UPEC strains are armed with virulence factors that specifically
target cytokine induction [113], a probiotic with the potential of increasing
the activation of NF-κB pathways would be a productive way of inhibiting
the colonisation and virulence properties of such pathogens.
MATTIAS KARLSSON I
45
Due to the NF-κB-modulating abilities of released products from
L. rhamnosus GR-1, we hypothesised that this bacterium could also
regulate the release of inflammatory cytokines, which was tested in Study
II. Accordingly, the increased activation of NF-κB in E. coli-challenged
cells after stimulation with live L. rhamnosus GR-1 was accompanied by
augmented levels of TNF. Although TNF is controlled by NF-κB, which is
activated by L. rhamnosus, Peña et al. [143] have reported decreased TNF
secretion in LPS-challenged macrophages after L. rhamnosus GG stimulation through a contact- and NF-κB-independent mechanism. Congruently,
Kim et al. [144] concluded that L. rhamnosus GR-1 and GG were potent
inhibitors of TNF in macrophages independently of NF-κB, through inhibition of MAP kinase pathways and production of anti-inflammatory
granulocyte colony-stimulating factor (GCSF). It was not determined in
Study II if the urinary bladder epithelial cells released GCSF, although
absence of this cytokine would partially explain why there were no reduced
levels of TNF in these cells. Given that lactobacilli can induce both NF-κB
and TNF, eventual GCSF production would be able to mask any NF-κBdependent TNF release. Although most studies have shown that lactobacilli
can actively down-regulate TNF, heat-killed L. casei and its LTA can
potently induce TNF secretion from immune cells as demonstrated by Kim
et al. [145] and Matsuguchi et al.[81]. Moreover, Castillo et al. [146] have
shown that mice continuously fed L. casei and subsequently challenged
with Salmonella produced augmented TNF release in immune cells isolated
from gut-associated lymphoid tissue, compared to mice not given L. casei.
Altogether, these data indicate that some lactobacilli can strengthen certain
aspects of the immune system.
While NF-κB and TNF were augmented by L. rhamnosus GR-1, the cells
in Study II did not respond with induction of IL-6 and CXCL8, two cytokines that are also under the influence of NF-κB. In fact, the levels of IL-6
and CXCL8 decreased when L. rhamnosus GR-1 was added to E. colichallenged cells. Nonetheless, unpublished data on IL6 and IL8 gene
regulation on T24 cells has revealed that L. rhamnosus GR-1 can potently
increase mRNA levels of both these cytokines. Given that both IL-6 and
CXCL8 are in part regulated by NF-κB [26, 27], a model where
L. rhamnosus GR-1 increases activation of NF-κB, which in turn initiates
transcription of TNF, IL6, and IL8 is appealing. To fit such a model, the
down-regulated protein levels of IL-6 and CXCL8 would be dependent on
other, post-transcriptional activities. This hypothesis is supported by
results from McCracken et al. [147] who found that a probiotic
L. plantarum strain reduced CXCL8 release from human intestinal cells
treated with TNF, although IL8 mRNA levels were increased.
46
I MATTIAS KARLSSON Ma et al. [148] have shown that L. rhamnosus efficiently inhibits
CXCL8 release in TNF-challenged human intestinal epithelial cells. There
was however no effect on CXCL8 when conditioned media containing
released products from L. rhamnosus was used, suggesting that regulation
of CXCL8 could depend on wall-anchored structures. Unfortunately, the
cytokine levels in Study II were not assayed after stimulation with heatkilled L. rhamnosus GR-1, though that would have revealed if the signals
governing NF-κB modulation were different from the ones controlling
cytokine release. Another aspect of the different responses after Lactobacillus stimulation was highlighted by Zhang et al., [149] who demonstrated that both live and dead L. rhamnosus GG were potent inhibitors of
TNF-induced CXCL8 secretion from human intestinal epithelial cells at
lower concentrations, while higher concentrations of the bacterium had the
opposite effect, leading to induction of CXCL8 release. Thus, the concentration of lactobacilli is imperative to the immunological outcome and
should be taken into account when discussing cellular responses to Lactobacillus stimulation as well as their use as immunomodulatory probiotics.
L. rhamnosus GR-1 modulates E. coli recognition in tissue cells
A major constituent of the UPEC outer membrane is LPS, which binds to
cells through TLR4 and thereby initiates an immunological reaction. Since
NF-κB is under TLR4 influence, it was natural to explore eventual effects
of LPS recognition as the cause of NF-κB augmentation by L. rhamnosus
GR-1. The T24 urothelial cells used in the study were found to express
TLR4, in line with other studies [150]. We continued to analyse the
involvement of this receptor by inhibiting LPS binding to TLR4 using polymyxin B (PMB). PMB avidly binds to LPS and blocks the eventual activation of TLR4, and thereby any further signalling. Adding PMB to the tissue
culture medium completely inhibited E. coli-dependent activation of NF-κB
as well as augmentation after the addition of lactobacilli (Figure 8), illustrating two important facts: (i) The NF-κB activation in T24 cells following
challenge with heat-killed E. coli was mainly due to LPS binding to TLR4
and (ii) the effect of L. rhamnosus GR-1 was dependent on signalling
through such receptor-ligand interaction.
MATTIAS KARLSSON I
47
Figure 8. Suppression of NF-κB augmentation by polymyxin B in urothelial cells
stimulated with E. coli and L. rhamnosus GR-1. P-values <0.05 compared to (a)
un-stimulated cells or (b) E. coli-challenged cells. Reproduced from [139].
Immunoassays (native immunoblot and confocal microscopy) showed that
co-stimulation of the urothelial cells with E. coli and L. rhamnosus resulted in increased levels of TLR4 protein thereby providing an explanation
for the augmented NF-κB activation. Although lactobacilli alone had a
marginal effect on TLR4 levels, the co-stimulation setting demonstrated a
significant increase in TLR4 compared to the other experimental groups.
Lactobacilli have previously been reported to modulate expression of
TLRs. Vizoso Pinto et al. [151] treated intestinal cells with L. rhamnosus
GG or an L. plantarum strain and found that protein levels of TLR2 and
TLR5 were increased by Lactobacillus treatment, while mRNA levels of
TLR4 remained unchanged. Castillo et al. [146] fed mice with L. casei, and
subsequently infected them with Salmonella and found significantly
augmented levels of TLR4 on isolated immune cells compared to cells from
mice not given lactobacilli.
TLR4 is crucial in the first attempts of localising invading microbes as
demonstrated by Schilling et al. [152] who exposed mice expressing defective TLR4 to UPEC. While normal mice responded with inflammation, the
urinary bladders of TLR4-defective mice showed no inflammation and
high numbers of UPEC in the urinary bladder. Accordingly, UPEC have
through evolution equipped themselves with various tools used to evade
host immunity, of which some act exclusively on the TLR pathway and
thereby inhibit inflammatory responses. Such strategies allow the microbes
to gain a foothold in the bladder, permitting them to divide and establish
48
I MATTIAS KARLSSON an infection. Increasing the levels of TLRs by administration of lactobacilli
could represent a productive way of facilitating pathogen recognition and
thereby reducing their virulence.
Released products from L. rhamnosus GR-1 are responsible for
NF-κB augmentation and contain putative immunomodulatory
substances
To further characterise the potentiated NF-κB activation during cochallenge of urothelial cells with heat-killed E. coli and live L. rhamnosus,
we continued by analysing different parts of the bacterium in study III. By
testing the effects of viable lactobacilli, released products, or heat-killed
bacteria, we could conclude that the highest retention of NF-κB augmentation was found in the released products from L. rhamnosus GR-1,
although heat-killed bacteria also augmented NF-κB activation. Because
both secreted products and heat-killed bacteria showed augmentation of
NF-κB, we continued to further analyse both of them. While isolation of
cell wall fragments using LiCl and trypsin treatment was unable to find the
cause of NF-κB augmentation, fractionation of the released products was
more fruitful.
Using a series of fractionations, based on protein mass, we were able to
produce five distinct fractions of released products from L. rhamnosus
GR-1. While two of the fractions were able to augment NF-κB activation
in urothelial cells during co-culture with E. coli, the other three had no
effect. Since it was the fractions obtained by molecular mass cut-off filters
with 100 and 50 kDa-sized pores (thus containing the largest proteins) that
showed activity, (Figure 9), the active protein (or protein complex) is likely
large in size and distributed in both of the two connecting fractions.
MATTIAS KARLSSON I
49
Figure 9. NF-κB activation in T24 urothelial cells following E. coli challenge (+)
and subsequent stimulation with released products (RP), or fractions (numbers
indicate molecular mass cut-off filter size) from L. rhamnosus GR-1 products. Pvalues <0.05 compared to (a) un-stimulated or (b) E. coli-challenged cells.
Two-dimensional gel electrophoresis of the released proteins from
L. rhamnosus GR-1 showed that numerous proteins were released into the
external environment during culture and subsequent isolation in PBS. Few
studies have previously described the Lactobacillus secretome. Although
L. rhamnosus GG is known to express a few proteins into the external
environment as described by Sánchez et al. [153], any in depth analysis of
these proteins has not been made. Zhu et al. [154] recently made a
thorough description of an L. plantarum proteome identifying more than
400 proteins, most of them being chaperones, proteins related to translation, or involved in carbohydrate metabolism. They also identified 22
secreted proteins, of which 11 were exclusively found in the cell-free
culture supernatant while not present inside the bacterial cell.
In study III, two-dimensional gel electrophoresis of the three largest
fractions (100 kDa, 50 kDa, and 30 kDa) showed that some proteins were
more abundant in the active fractions compared to fractions that failed to
augment NF-κB activation. A total number of 10 proteins were identified
using mass spectrometry and subsequent matching in protein databases
(Table 2). Two protein spots that were highly abundant in all three
fractions were used as reference points in order to superimpose all gels and
thereby select the remaining candidate spots for identification. Once gels
were properly superimposed (aligned), eight more spots were selected
based on their high abundance in the two active fractions giving NF-κB
50
I MATTIAS KARLSSON augmentation, and similarly low amount of the corresponding spots in the
neighbouring fraction that did not augment NF-κB activation.
Table 2. Identified proteins from two-dimensional gel electrophoresis and their
predicted secretion based on classical or non-classical secretion signals.
Protein
Secreted §
Chaperonin GroEL
No
Elongation factor Tu
No
Enolase
No
FKBP-type peptidyl-propyl cis-trans isomerase
Yes
Fructose/tagatose bisphosphate aldolase
No
Glyceraldehyde-3-phosphate dehydrogenase*
No
L-lactate dehydrogenase
No
NLP/P60 family protein
Yes
Phosphoglycerate kinase
No
Triosephosphate isomerase*
No
§
Predicted secretion pattern.
* Reference proteins found in all three fractions.
Out of the ten identified proteins, only two of them were predicted to
actively secrete into the environment, namely FKBP-type peptidyl-propyl
cis-trans isomerase and the NLP/P60 family protein. The rest of the
proteins were assumed to be cytosolic. A number of the proteins identified
in Study III were also found by Zhu et al. [154] to be released by
L. plantarum, despite their lack of proper secretion signals. Functionally, a
majority of the proteins (six out of ten) were important members of
glycolysis and lactic acid fermentation.
The NLP/P60 family protein was predominantly found in the highly
active 50 kDa fraction. This protein has a 40 % sequence similarity to a
secreted protein identified in L. rhamnosus GG by Sánchez et al. [153].
Both of these proteins share a domain with an important role in Listeria
virulence, especially the bacterium’s ability to invade human intestinal cells
[155]. Moreover, the NLP/P60 protein identified in L. rhamnosus GR-1
released products demonstrated over 40 % homology with cytoprotective
p40 and p75 proteins from L. rhamnosus GG, suggesting that this protein
could share cytoprotective functions found in p40 and p75. Although these
proteins have an unclear effect on NF-κB, their impact on MAP kinases has
already been demonstrated by Seth et al. [85].
MATTIAS KARLSSON I
51
Two of the most interesting proteins identified in the active fractions
were EF-Tu and GroEL. EF-Tu is a key protein in the translation of mRNA
transcripts to proteins inside the cell but is also found attached to the cell
wall or released from bacteria, and was predominantly found in the two
active fractions. Granato et al. [87] isolated EF-Tu from L. johnsonii and
found that it efficiently attached to human intestinal epithelial cells
suggesting that this protein is important in adhesion to the mucosa.
Furthermore, they found that binding of EF-Tu resulted in increased
CXCL8 release from the intestinal epithelial cells thus demonstrating its
immunomodulatory action. Chaperonin GroEL is a major constituent of
the bacterial cytoplasm where it aids in protein folding. In study III, GroEL
was predominantly found in the 50 kDa fraction, although it was also
present in the 100 kDa fraction. Though GroEL is predicted to be a cytosolic protein lacking any known secretion signal, Bergonzelli et al. [86]
found that GroEL from L. johnsonii was both anchored to the cell wall of
bacteria, and released in culture. Moreover, this isolated GroEL potently
induced secretion of CXCL8 in human intestinal epithelial cells and
macrophages.
Since both EF-Tu and GroEL are proteins sensitive to heat, heat-killing
lactobacilli might miss the eventual effects by proteins that are found in the
cytosol and on the surface of cells. By heat-killing L. rhamnosus GR-1 in
Study II and using isolated released products it was concluded that the
effects on NF-κB were due to a released product from the bacteria.
However, by choosing a different method of analysing effects of non-viable
lactobacilli (e.g. by antibiotics or lethal irradiation) that do not affect heatlabile components, the results might have differed and pointed towards a
cytosolic or surface-bound Lactobacillus protein, that was also being
released from the cell.
The effects of L. rhamnosus GR-1 on immune cells are complex
Much of the work in this thesis has focused on urothelial cells and their
elicited innate immune responses when facing lactobacilli. Although
urinary epithelium mainly consists of epithelial cells, other cell types are
present in both uninfected and infected tissue. Macrophages are relatively
abundant in the bladder, and increases after infection with UPEC, along
with granulocytes and dendritic cells [156]. The phagocytic and antigenpresenting abilities of immune cells are much needed at the bladder-lumen
interface. Since previous studies had showed that L. rhamnosus GR-1
exhibited immunomodulatory actions on urothelial cells (Study I-III), it
52
I MATTIAS KARLSSON was of interest to investigate the responses of macrophages to probiotic
lactobacilli.
To test the cytokine-modulating properties of L. rhamnosus GR-1, a
model using two cell lines was devised. By using a transwell insert, fitted
into the original well, two cell types could be grown without any physical
contact. Media and small molecules were able to pass through 0.2 µmsized pores throughout the membrane on which the upper cell type was
grown. Large particles such as bacteria added to the upper well (insert)
could however not get through because of their size. To simulate the way
in which E. coli have contact with the cells of the urinary bladder, we
cultured urothelial cells (5637 bladder cell line) in the top well at near confluency while macrophage-like cells (differentiated THP-1 cells) were
grown in the bottom well.
Co-culturing the two cell lines together in this form did not significantly
alter the cytokines tested, except for IL-6, which was increased in the
macrophage-like cells. We continued by adding heat-killed E. coli to the
upper well with urothelial cells, to bring about an increase in the proinflammatory cytokines. Addition of bacteria increased cytokine release
from both cell types, even though there was no direct contact between the
bacteria and the macrophage-like cells. Likely, small pieces of
immunostimulatory nature (e.g. LPS) could pass through gaps in the cell
layer and cross the semi-permeable membrane, docking onto PRRs present
on the macrophage-like cells.
One of the main purposes with Study IV was to evaluate differences in
cytokine release from macrophage-like cells directly or indirectly stimulated with L. rhamnosus GR-1. Although lactobacilli had a great impact on
cytokine release during direct treatment of macrophage-like cells, these
effects were markedly reduced when bacteria did not have contact with the
macrophage-like cells and none of the cytokines were altered by lactobacilli
treatment alone during indirect treatment.
Direct treatment with L. rhamnosus potently increased the levels of IL1β. Hayes and Zoon [157] have previously reported that IL-1β enhances
TNF expression in macrophages exposed to LPS. However, the increase in
IL-1β during direct treatment of cells with lactobacilli was not accompanied by TNF increase. Although L. rhamnosus GR-1 could augment TNF
release from T24 cells (Study II), the role of IL-1β was unfortunately not
determined. During indirect treatment with lactobacilli in Study IV,
macrophage-like cells did not show any increase in IL-1β, although TNF
levels were augmented. The corresponding effects on urothelial cells were
not significant. It should however be noted that the 5637 cells in Study IV
MATTIAS KARLSSON I
53
released very minute amounts of TNF compared to T24 cells that were
used in Study I-III.
L. rhamnosus GR-1 has previously been shown to down-regulate TNF
in macrophages by Kim et al. [144]. Moreover, Peña et al. [143] have
shown the same thing for L. rhamnosus GG. In accordance with those
studies, direct treatment substantially decreased release of TNF from
E. coli-challenged macrophage-like cells. However, physical separation of
lactobacilli from the macrophages led to augmentation of TNF release,
suggesting this trait is in part governed by factors that do not penetrate the
semi-permeable membrane. Ménard et al. [158] have studied the effects of
lactic acid bacteria on LPS-induced TNF secretion in macrophages and
found that conditioned media lacking of any viable bacteria were potent
suppressors of TNF release, not dependent on contact between bacteria
and macrophages.
The most important discoveries in Study IV were related to IL-6 and
CXCL8 regulation and demonstrate how differently these two cytokines
are influenced by L. rhamnosus GR-1. The high levels of IL-6 found after
E. coli challenge in both cell types and culture conditions were significantly
decreased after L. rhamnosus GR-1 treatment in both cell lines and in a
contact-independent fashion. Clearly, Lactobacillus material was transferred through the pores of the semi-permeable membrane separating the
two cell types, telling us that the nature of the IL-6 regulatory substance is
also released. Miettinen et al. [159] reported that lactobacilli, including
L. rhamnosus GG, are potent activators of IL-6 in macrophages, however,
only when killed. Such results indicate that secreted substances likely mask
effects of immunostimulatory wall fragments. Unfortunately, the authors
did not investigate the effects of L. rhamnosus in cells challenged with
E. coli or other bacteria.
As for IL-6, CXCL8 secretion from macrophage-like cells challenged
with E. coli was efficiently quelled by direct treatment with L. rhamnosus
GR-1. The effects on urothelial cells were comparable. However, during
co-culture of tissue cells, when lactobacilli did not have direct contact with
the macrophage-like cells, the CXCL8 levels were not altered but remained
high. The most likely explanation for these results is that some of the
factors governing CXCL8 regulation by L. rhamnosus are dependent on
physical contact between lactobacilli and the eukaryotic cells or CXCL8
directly. Such contact-dependent immunomodulatory effects of lactobacilli
have previously been established. Ma et al. [148] have shown that
L. rhamnosus can efficiently inhibit CXCL8 release in TNF-treated human
intestinal epithelial cells, although conditioned L. rhamnosus media was
ineffective. Others have also clearly indicated that immunomodulatory
54
I MATTIAS KARLSSON actions of lactobacilli can solely rely on wall-anchored structures. Kim et
al. [145] created cell wall mutants of an L. casei strain. While the wild type
Lactobacillus strain was a strong activator of innate immunity in mice, the
cell wall mutant had no effect on cytokine expression.
The most likely explanation for the absent regulation of CXCL8 in coculture is that the phenomenon is guided by cell wall-anchored structures.
However, it cannot be ruled out that the urothelial cells in the upper well
secrete substances that could influence the cytokine responses in macrophage-like cells. Haller et al. [160] have demonstrated that there is crosstalk between intestinal epithelial cells and immune cells in a gut model,
affecting cytokine secretion from cells after E. coli and Lactobacillus
challenge. Such cross-talk might also be important in a corresponding
model of the bladder comprised of urothelial and macrophage-like cells as
in Study IV.
A necessary feedback of pro-inflammatory cytokine production and
signs of inflammation is the subsequent production of factors that contribute to resolution of inflammation. One of these factors is the IL-6
receptor (IL-6R), produced in two main forms: membrane bound (mIL-6R)
and soluble (sIL-6R) [161]. Interestingly, E. coli-challenged macrophagelike cells secreted less sIL-6R compared to un-stimulated cells. Moreover,
L. rhamnosus GR-1 treatment counteracted the decreased release, a
completely novel finding that signifies that factors from this microbe could
be able to promote healing after infection, although such hypotheses would
have to be tested in further studies.
Lactobacilli demonstrate inter-species variability in urothelial
cell CXCL8 modulation
Lactobacillus is a large genus within the group of lactic acid bacteria.
There are over 100 species and most of them have a notable number of
strains identified or even sequenced. Because of the great genetic variation,
we decided to test differences in immune regulation between different
Lactobacillus strains in Study V.
The chemokine CXCL8 was chosen as a marker for two reasons. Firstly,
CXCL8 is one of the most important cytokines secreted by the urothelium
during infection. Secondly, we had previously observed that this cytokine
was readily released from the urothelial cells (5637 cell line) during E. coli
challenge. The influence of lactobacilli on CXCL8 release in urothelial cells
was evaluated in two different conditions. Resting and E. coli-challenged
cells were subjected to different Lactobacillus species. The screening of
lactobacilli showed a heterogeneous effect on CXCL8 release from the
MATTIAS KARLSSON I
55
urothelial cells. While L. crispatus, L. helveticus, L. reuteri, L. acidophilus
and L. sakei induced the release of CXCL8 from urothelial cells, L. casei,
L. paracasei, L. johnsonii, L. delbrueckii, and L. rhamnosus had the opposite effect. The ability to reduce CXCL8 was particularly strong when cells
were concomitantly challenged with E. coli.
Christensen et al. [162] have previously described that Lactobacillus
species differently influence release of cytokines in immune cells. Although
the authors did not study the effects on CXCL8, they reported that lethally
irradiated L. reuteri, L. plantarum, L. casei, L. fermentum, and
L. johnsonii were able to differently influence release of IL-6, IL-10, IL-12
and TNF from dendritic cells. Similar studies on epithelial cells from
urinary epithelial cells are lacking, although Vizoso Pinto et al. [151] have
demonstrated that L. rhamnosus GG and an L. plantarum strain can
augment CXCL8 release in intestinal epithelial cells after Salmonella
infection or TNF challenge. In sharp contrast, we observed that
L. rhamnosus GR-1 was a strong suppressor of CXCL8 after E. coli
challenge (Study II and IV). If these discrepancies reflect physiological
differences between intestinal and urinary bladder cells, or are dependent
on the exact stimulus (TNF, Salmonella, or E. coli) is not known.
However, Donato et al. [163] and Zhang et al. [149] have shown that
L. rhamnosus GG is able to reduce CXCL8 levels in TNF-stimulated
intestinal cells. Furthermore, Donato et al. [163] demonstrated that while
L. rhamnosus GG effectively reduced CXCL8 secretion, an L. plantarum
strain showed no effect on this cytokine. Conversely, McCracken et al.
[147] found that another L. plantarum strain strongly inhibited TNFdependent CXCL8 release from intestinal cells, highlighting immunomodulatory differences between strains within the same species.
One of the strongest CXCL8 motivators in Study V was L. helveticus.
GroEL from L. helveticus has been reported to induce CXCL8 in both
epithelial cells and macrophages by Bergonzelli et al. [86], although the
effects on CXCL8 were not tested using whole viable cells. Moreover, we
found that L. sakei promoted the release of CXCL8 from urothelial cells.
Correspondingly, Haller et al. [160] have previously demonstrated that
L. sakei but not L. johnsonii can induce transcription of IL8 in intestinal
epithelial cells. Accordingly, L. johnsonii did not induce CXCL8 release in
Study V. Although the mechanisms resulting in the regulation of CXCL8
was not determined, LTA is a likely target for future research. Vidal et al.
[164] have demonstrated that isolated LTA from L. johnsonii and
L. acidophilus can efficiently inhibit LPS-induced CXCL8 secretion from
intestinal epithelial cells. Moreover, p40 and p75 homologues are
expressed by L. rhamnosus, L. casei, and L. paracasei [165], who all
56
I MATTIAS KARLSSON reduced CXCL8 release in Study V. Since these proteins are known
modulators of immune responses, they could possibly be involved in this
CXCL8 response.
Two possible routes affecting the amount of CXCL8 in the cell culture
supernatants were tested in Study V. First, it was concluded that the downregulated CXCL8 secretion by L. delbrueckii was not dependent on
degradation of CXCL8 in the extracellular milieu. Bacterial proteases that
degrade cytokines in the extracellular space have previously been described
for other microbes [166], although not from lactobacilli. Additional studies
on other lactobacilli that reduce CXCL8 are however motivated since there
are likely several mechanisms that control the reduction of CXCL8 and
other cytokines, some of them including physical destruction of the
protein. A substance from lactobacilli with the above function would be
able to influence the course of an immune response without directly acting
on the individual cells within the tissue. Second, we could not correlate the
effects on CXCL8 protein levels to transcriptional regulation. Although
selected lactobacilli could alter IL8 transcription, the results did not match
the trend from CXCL8 protein release. Similarly, McCracken et al. [147]
found that an L. plantarum strain able to reduce CXCL8 release from
TNF-challenged intestinal cells increased IL8 mRNA levels, demonstrating
that the Lactobacillus effects on this cytokine is possibly not dependent on
transcriptional regulation. Moreover, this is in line with previous results on
L. rhamnosus GR-1 (Study IV) showing reduced CXCL8 release from
E. coli-challenged 5637 urothelial cells, yet increased IL8 transcription.
In order to expose similarities and differences in CXCL8 regulation from
a phylogenetic perspective, a dendogram was constructed using 16S ribosomal RNA sequences also carrying information from screening of CXCL8
release after Lactobacillus challenge. The main results affirmed that lactobacilli with similar effects on CXCL8 release were also phylogenetically
grouped very closely together (Figure 10).
MATTIAS KARLSSON I
57
Figure 10. Dendogram constructed from 16S rRNA of species used in CXCL8
release screening. B. subtilis was used as an outgroup during tree assembly. (↓)
indicate reduced CXCL8 release from un-stimulated and E. coli-challenged cells
(except L. johnsonii), while (↑) indicate an increase in CXCL8 release from unstimulated cells. Bar represents evolutionary distance.
L. rhamnosus, L. casei, and L. paracasei that reduced CXCL8 release
from urothelial cells are all genetically very similar. The fact that they also
shared the CXCL8-reducing phenotype was therefore very interesting.
Analogous results were obtained for L. acidophilus, L. crispatus, and
L. helveticus that increased CXCL8 secretion and demonstrated close
genetic relationship. Although no in-depth analysis on the origin of these
traits was performed in Study V, the findings from these two diametrically
opposite groups can be used to speculate on whether these traits are
homologous or homoplasious.
58
I MATTIAS KARLSSON CONCLUSIONS AND FUTURE PERSPECTIVES
Conclusions
The main findings and conclusions of this thesis are summarised as
follows:
• Live L. rhamnosus GR-1 and its released products marginally
affected NF-κB activation in resting urothelial cells, while they
augmented NF-κB activation in E. coli-challenged cells (Study I and
II).
• NF-κB augmentation in urothelial cells correlated with increased
levels of TNF, although CXCL8 and IL-6 were decreased (Study II).
• NF-κB augmentation by L. rhamnosus GR-1 was dependent
on enhanced recognition of LPS by the cells, through TLR4 (Study
II).
• L. rhamnosus GR-1 secreted numerous proteins into the extracellular environment during growth, some of which have described
immunomodulatory actions (Study III).
• L. rhamnosus GR-1 caused release of pro-inflammatory IL-1β in
urothelial cells and macrophage-like cells (Study IV).
• L. rhamnosus GR-1 regulated cytokine release from macrophagelike cells, in both contact-dependent (CXCL8) and contactindependent (IL-6) manners (Study IV).
• Lactobacilli could differently influence CXCL8 release from urothelial cells, depending on the species used (Study V).
• Lactobacillus species with analogous effects on CXCL8 release
exhibited close evolutionary relationship (Study V).
Future perspectives
The primary aims of this thesis were to analyse the immunomodulatory
role of lactobacilli, highlighting the effects on epithelial cells of the urinary
system. L. rhamnosus GR-1 is one of the most studied urogenital probiotics and therefore, most of the studies focused on this particular strain.
Although apparent that lactobacilli have a great potential for modulating
mucosal immunity, whether or not these attributes are essential for probiotic effects is currently unknown. It is therefore necessary that future
analyses on probiotics continue to dwell into the immunomodulatory
mechanisms of substances and stringently deconstruct and assess their
contribution to the function of probiotics. In study II, one important
MATTIAS KARLSSON I
59
receptor, TLR4, was identified to be involved in the Lactobacillusdependent NF-κB modulation. There are several additional factors
controlling responses towards pathogenic microbes, including other TLRs,
as well as cytokine and chemokine receptors.
Isolating immunoactive substances from probiotic bacteria that contribute to their probiotic effects have clear benefits. Isolated substances
pose no risk in terms of resistance transfer, or infection in immunocompromised individuals when used therapeutically. Moreover, there are
general concerns surrounding the administration of viable bacteria to
sterile or near-sterile loci such as the urinary bladder or urethra due to the
possible risk of adverse immune responses. Isolated material with specific
immunological targets would diminish the risk of such side effects.
Although the work in Study III identified several putative immunomodulatory proteins in the released products from L. rhamnosus GR-1,
these proteins were not isolated or synthesised and are therefore, so far
only suitable candidates. Generation of specific mutations in these proteins
would offer unique opportunities to study their role in modulation of
cellular innate immunity.
Given immunomodulation is an important trait and that most probiotics
today are selected based on their ability to influence growth, pathogenicity,
and adhesion of pathogens, screening programmes directed to assay
immunomodulatory features would allow scientist to identify immunomodulatory fulcra and select probiotics with new and unique features.
Screening of twelve Lactobacillus strains in Study V showed that lactobacilli with high genetic similarity also exhibited the same effect on
CXCL8 release from urothelial cells, suggesting that homologous traits in
these lactobacilli were responsible for the outcome. Additional studies
analysing surface structures and released products from the different
Lactobacillus groups could uncover important immunomodulatory factors,
including responses based on non-secreted products.
60
I MATTIAS KARLSSON ACKNOWLEDGEMENTS
I would like to thank a number of people who have made this thesis possible.
My primary supervisor, Jana Jass, for critical reading of manuscripts and
the thesis, good suggestions, and countless interesting discussions.
My assisting supervisor, Robert Brummer, for critical reading of the thesis
as well as Study III, IV, and V.
Per-Erik Olsson, for good suggestions on how to improve Study V, and for
the collaboration on Study III and IV.
Hazem Khalaf, for the collaboration on the Lactobacillus fractionation in
Study III and good discussions on NF-κB and immunology.
Nikolai Scherbak, for the collaboration on Study I-IV and your support.
Hanan Abuabaid, for interesting discussions concerning regulation of immune responses in macrophages, as well as the collaboration on Study IV.
Simon Lam, for the collaboration on Study I (and a lot of other things).
Gregor Reid, for collaboration on Study II.
Katarina Larsson and Ingrid Lindh, for well-needed input on the Swedish
summary!
Peer-reviewers and editors, for their good suggestions on how to improve
the studies.
My family and friends, for your never-ending support and for always
believing in me!
The work behind this thesis has been supported by:
• Carl Tryggers Stiftelse för Vetenskaplig Forskning
• Sparbanksstiftelsen Nya
• Stiftelsen för kunskaps- och kompetensutveckling (KK-stiftelsen)
• Helge Ax:son Johnsons Stiftelse.
MATTIAS KARLSSON I
61
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SVENSK SAMMANFATTNING
Inledning
Den här avhandlingen avser belysa ett område där forskningen ännu är
mycket begränsad, nämligen urinvägarnas reaktioner på laktobaciller.
Studierna i den här avhandlingen har analyserat förändringar i de direkta
immunsvaren (främst aktivering av NF-κB och cytokinproduktion) från
urotelceller efter kontakt med laktobaciller och urinvägspatogener. Fokus
har huvudsakligen legat på användandet av en av de mest studerade urogenitala probiotiska bakterierna, Lactobacillus rhamnosus GR-1, även om
andra laktobaciller också har testats.
Probiotika är mikroorganismer som främjar hälsan och används främst
vid behandling samt förebyggande av gastrointestinala besvär. Under
senare år har forskning även visat att vissa probiotika kan skydda såväl
mot en rad infektioner som mot allergier. De flesta probiotiska mikroorganismerna är laktobaciller: stavformade bakterier som vanligtvis
koloniserar människan. De återfinns främst i den urogenitala mukosan
(vaginan), men koloniserar i viss utsträckning även våra tarmar. Det har
länge antagits att våra urinvägar i normalfallet är sterila, men nya
forskningsresultat har visat att laktobaciller i urinvägarna är ett normalt
fynd. De eventuella följderna av kolonisering med laktobaciller är
emellertid inte klarlagda. Det är dock känt att laktobaciller alstrar rikliga
mängder mjölksyra under tillväxt, som sänker pH-värdet och drastiskt
försvårar kolonisering av sjukdomsalstrande bakterier. Många laktobaciller har även möjligheten att producera väteperoxid eller specifika
proteiner som är giftiga för andra bakterier. Utöver dessa direkta
mekanismer riktade mot andra mikroorganismer har laktobaciller visat sig
kunna påverka människors immunförsvar.
Sjukdomsframkallande (patogena) mikroorganismer (framförallt vissa
stammar av Escherichia coli) i urinvägarna leder ibland till infektion i urinrör eller urinblåsa. Dessa urinvägspatogener, som ofta härstammar från
personens egen tarmflora, bär många specifika vidhäftningsmolekyler som
bidrar till bakteriens koloniseringsmöjligheter genom att underlätta vidhäftning till de urotelceller som utgör urinvägarnas slemhinnor. Utöver
vidhäftningsmolekyler uttrycker många urinvägspatogener substanser som
hämmar normala immunsvar, i hopp om att undkomma eliminering.
En avsevärd del av normala immunsvar under infektioner är beroende
av transkriptionsfaktorn NF-κB. Detta proteinkomplex aktiveras snabbt då
celler får kontakt med mikroorganismer såsom urinvägspatogener.
Särskilda strukturer hos bakterier (exempelvis E. coli) binder till
MATTIAS KARLSSON I
79
mottagarmolekyler på värdcellernas yta och orsakar en reaktion som
mynnar ut i produktionen av ämnen som initierar immunsvar, bland annat
cytokiner. Detta sker i immunceller såväl som i slemhinnornas epitelceller.
Cytokiner utsöndrade från slemhinneceller (till exempel urotelceller i urinvägarna) bidrar i sin tur till en lokal inflammation med tillströmning av en
avsevärd mängd vita blodkroppar till det infekterade området, vilket i de
flesta fall leder till att den sjukdomsframkallande mikroorganismen
elimineras.
Urinvägspatogener som lyckas fästa till slemhinnan i urinvägarna och
undkomma immunförsvaret kan orsaka en urinvägsinfektion (UVI).
Ungefär hälften av alla kvinnor har minst en UVI under sin livstid, vilket
medför att UVI är en av de vanligaste bakteriella infektionerna. En
betydande andel av de som genomgått en UVI drabbas av recidiv, med
infektioner som återkommer flera gånger per år och är svåra att förebygga.
Låga doser av antibiotika under en längre tid kan användas i förebyggande
syfte, men en ökad antibiotikaresistens hos urinvägspatogener och många
biverkningar motiverar sökandet efter alternativa behandlingar.
Ett fåtal laktobaciller har testats för att förhindra återkommande UVI
hos kvinnor, med i stor utsträckning positiva resultat. En av dessa
stammar, L. rhamnosus GR-1, isolerat från urinröret hos en kvinna, är en
av de mest studerade urogenitala probiotiska bakterierna. Denna bakterie
säljs som en produkt innehållande två laktobaciller: L. rhamnosus GR-1
och L. reuteri RC-14. Tillsammans har dessa bakterier visat sig kunna
minska frekvensen av UVI hos kvinnor som vanligtvis har återkommande
infektioner. Förutom att förhindra tillväxt och vidhäftning av urinvägspatogener har probiotiska mikroorganismers inflytande över immunsvar
föreslagits som en viktig komponent i deras probiotiska verkan.
Förstärkta immunsvar av L. rhamnosus GR-1
Eftersom det inte var känt sedan tidigare hur urotelceller påverkas av
laktobaciller utfördes ett initialt test där produkter som utsöndrats från
probiotiska L. rhamnosus GR-1 och L. reuteri RC-14 användes för att
stimulera urotelceller i cellodling. Ingen av de två bakterierna utsöndrade
produkter som hade någon avsevärd effekt på NF-κB, ett av de största
proteinkomplexen som styr immunsvar i dessa celler. Urotelceller som
utsattes för E. coli (den viktigaste urinvägspatogenen) reagerade dock med
en kraftfull aktivering av NF-κB, vilket visade att cellernas immunologiska
svar påverkades av dessa bakterier. Även om produkter från L. rhamnosus
GR-1 inte hade någon större effekt på NF-κB hos annars vilande celler
kunde de potentiera aktiveringen av NF-κB hos E. coli-stimulerade celler.
Laktobacillerna kunde med andra ord förstärka immunsvaret mot E. coli.
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I MATTIAS KARLSSON Produktionen av TNF, en viktig inflammatorisk cytokin som normalt
uttrycks med hjälp av NF-κB, potentierades också av L. rhamnosus GR-1,
medan utsöndrandet av två andra cytokiner (IL-6 och CXCL8) minskade.
Alltså, medan laktobacillerna ökade produktionen av TNF hos E. colistimulerade celler, gick nivåerna av IL-6 och CXCL8 ner, vilket
exemplifierar den stora komplexitet som styr immunsvar hos urotelceller.
TLR4 är en central mottagarmolekyl hos urotelceller och binder normalt
sett lipopolysackarider som finns på ytan hos E. coli, vilket i sin tur leder
till aktivering av bland annat NF-κB. När bindning av lipopolysackarider
till TLR4 blockerades med hjälp av en kemikalie inhiberades också
effekten av L. rhamnosus GR-1 på NF-κB. Mängden TLR4 på urotelcellerna visade sig också öka av stimulering med L. rhamnosus GR-1.
Potentieringen av NF-κB kan således bero på att cellerna, genom ett ökat
antal mottagarmolekyler (TLR4) på sin yta, lättare reagerar på potentiellt
farliga mikroorganismer i sin omgivning. Det är möjligt att denna effekt
representerar en viktig probiotisk mekanism hos L. rhamnosus GR-1.
Sekreterade substanser bidrar aktivt till förändrade immunsvar
För att vidare testa möjligheterna hos L. rhamnosus GR-1 att påverka
immunsvar hos urotelceller undersöktes hur levande respektive värmedödade laktobaciller påverkade NF-κB. Medan levande laktobaciller hade
en tydlig inverkan på NF-κB, var effekten hos värmedödade bakterier
starkt begränsad. Eftersom effekten var mest påtaglig när bakterierna var
levande indikerade detta att substanser som producerades under tillväxt
var betydande för effekten på NF-κB. Vid tillväxt utsöndrar bakterier en
mängd olika produkter. De utsöndrade produkterna från L. rhamnosus
GR-1 delades upp med avseende på deras storlek i totalt fem olika
fraktioner. Två av dessa fraktioner hade möjligheten att potentiera
aktiveringen av NF-κB i E. coli-stimulerade urotelceller på liknande sätt
som icke-fraktionerade produkter. Genom att jämföra de proteiner som
ingick i de aktiva fraktionerna med de proteiner som återfanns i fraktioner
som inte påverkade NF-κB identifierades flera proteiner med immunomodulerande verkan, bland annat chaperonet GroEL, elongeringsfaktorn
Tu samt ett cellväggshydrolyserande protein tillhörande NLP/P60-familjen.
Dessa proteiner har i vetenskapliga studier tillskrivits immunomodulerande
egenskaper, men har tidigare inte identifierats hos L. rhamnosus GR-1.
Studier på vita blodkroppar ger mer information
Även om urotelceller är den mest förekommande celltypen i urinvägarna så
finns det också där, likt i all annan vävnad, vita blodkroppar insprängda i
MATTIAS KARLSSON I
81
de underliggande cellagren. Makrofager är en av de första vita blodkropparna som aktiveras i ett infekterat vävnadsområde. Efter aktivering
(bland anat av lipopolysackarider från E. coli) utsöndrar dessa celler stora
mängder cytokiner. L. rhamnosus GR-1 påverkade märkbart utsöndrandet
av olika inflammatoriska cytokiner (TNF, IL-1 beta, IL-6 och CXCL8)
från makrofager i cellodling. Tydligast var ökningen av proinflammatoriskt IL-1 beta.
För att studera kontakt-beroende och kontakt-oberoende effekter
separerades laktobaciller från makrofagerna med hjälp av ett semipermeabelt membran, täckt med urotelceller. När laktobaciller separerades
från makrofagerna under stimulering visade det sig att effekterna på
cytokinproduktion inte längre var synliga. Som tidigare observerats hos
urotelceller så minskade produktionen av IL-6 och CXCL8 hos E. colistimulerade makrofager signifikant när laktobaciller tillsattes. De makrofager som blev indirekt stimulerade (det vill säga att laktobacillerna var
separerade från makrofagerna) hade fortsatt höga nivåer av CXCL8 men
en klar minskning av IL-6. Ett sådant fynd antyder att de faktorer hos laktobacillerna som styr immunmodulering är flera, och att deras
verkningsmekanism skiljer sig betydligt åt. Då laktobacillernas effekt på
sekretion av CXCL8 endast var synlig när bakterierna fick kontakt med
cellerna
indikerar det att effekten är beroende av en fysisk kontakt mellan laktobaciller och makrofager. Jämförelsevis tyder låga nivåer av IL-6 i båda
stimuleringsgrupperna på att sekreterade substanser från laktobaciller
transporteras genom det semipermeabla membranet och tillåts därigenom
förändra sekretionen av IL-6.
TNF-svaret hos E. coli-stimulerade makrofager blev väsentligt hämmat
efter tillförsel av L. rhamnosus GR-1 direkt på cellerna, även om de
indirekt stimulerade makrofagerna gav ett potentierat TNF-svar likt det
som tidigare observerats hos urotelceller. Dessa resultat kan sammantaget
sägas uttrycka den enorma komplexitet som konstituerar produktionen av
olika cytokiner i både urotelceller och vita blodkroppar såsom makrofager.
Tillika verkar signalerna som styr cytokinsekretion vara beroende av olika
bakteriella komponenter. Medan några effekter krävde kontakt mellan
laktobaciller och de eukaryota cellerna baserades andra signaler på utsöndrade produkter som kunde passera det semipermeabla membranet.
Stora variationer mellan laktobaciller
CXCL8 sekreteras av slemhinneceller vid infektioner för att attrahera vita
blodkroppar till det skadade området. Fel i sekretionen av CXCL8 är
kopplat till vissa sjukdomstillstånd, bland annat UVI. Laktobaciller är ett
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I MATTIAS KARLSSON stort släkte med en enorm genetisk variation, vilket också kan innebära att
effekterna av stimulering med olika laktobaciller kan skilja sig åt. För att
testa skillnader i CXCL8-reglerande effekter hos laktobaciller valdes tolv
laktobaciller ut från olika arter inom släktet, vilka sedan användes för att
stimulera urotelceller. För att få en klar bild av laktobacillernas samlade
effekter, även under infektionsliknande förhållanden där sekretion av
CXCL8 är viktigt, stimulerades även celler som redan utsatts för E. coli. En
majoritet av laktobacillerna förändrade på något sätt sekretionen av
CXCL8, och det skedde i båda riktningarna. Således kunde laktobacillerna
ha en direkt motsatt effekt på CXCL8, beroende på vilken art som
användes. Laktobaciller med likartad inverkan på CXCL8 visade sig
dessutom ha ett starkt evolutionärt släktskap, vilket kan tyda på att
gemensamma strukturer i de olika arterna med effekter på CXCL8
bevarats under evolutionen.
Utvalda arter med effekter på CXCL8 användes vidare för att studera
eventuella mekanismer ansvariga för denna reglering av CXCL8. De
testade bakterierna visade sig inte påverka CXCL8 extracellulärt, det vill
säga laktobacillerna utsöndrade inga substanser som kunde förändra
mängden CXCL8 efter att den producerats av cellerna. En del laktobaciller
visade en tendens till att influera uttryck av genen för CXCL8, men denna
reglering av genen för CXCL8 sammanföll inte med vad som tidigare
observerats på proteinnivå. Eftersom laktobacillerna inte hade någon
avsevärd effekt på varken proteinet CXCL8, eller på dess uttryck (genen),
är det möjligt att bakterierna i stället kan manipulera hur proteinet efter
tillverkning sekreteras av cellen.
Nya möjligheter
Många människor använder i dag probiotika för att lindra eller förebygga
sjukdomar, främst gastrointestinala besvär. Experimentell forskning har
dock visat att det finns stora möjligheter när det gäller användandet av
probiotika, även för andra åkommor såsom UVI och allergier. Antalet
studier inom området är emellertid än så länge mycket begränsat.
Selektion av probiotiska mikroorganismer har traditionellt skett i labbmiljö, främst baserat på hur väl mikroorganismerna kan fästa till slemhinneceller samt på produktionen av antibakteriella substanser, som kan
hindra kolonisering med sjukdomsframkallande mikroorganismer. Efter en
sådan karaktärisering har dessa bakterier utvärderats i kliniska studier,
ibland med stor framgång. Att analysera probiotiska mikroorganismers
möjlighet att förändra immunsvar hos en värd representerar en ny syn på
hur probiotiska mikroorganismer agerar. Den här avhandlingen har
analyserat bakteriella produkter som påverkar immunsvar i celler från
MATTIAS KARLSSON I
83
urinvägarna, samt föreslagit mekanistiska förklaringar till dessa. För att
det ska vara möjligt att utnyttja de inneboende immunologiska krafterna
hos probiotiska bakterier måste vi lära oss mer om på vilka sätt dessa kan
förändra immunsvar, vilka de önskvärda svaren är och vid vilka sjukdomstillstånd en viss effekt är önskvärd. En komplett bild av laktobacillernas
effekter möjliggör att denna kunskap kan användas vid val av probiotisk
behandling och för att hitta nya bakterier med önskvärda effekter. Om de
specifika substanserna från bakterierna kan identifieras medför den nya
gentekniken stora möjligheter att skapa probiotiska hybrider med goda
egenskaper från olika mikroorganismer. I stället för att tillverka produkter
som innehåller en blandning av olika probiotiska mikroorganismer kan
alla goda egenskaper från dessa samlas i en enda produkt som bättre kan
främja hälsa än var och en av dessa för sig.
84
I MATTIAS KARLSSON Publications in the series
Örebro Studies in Life Science
1. Kalbina, Irina (2005). The molecular mechanisms behind perception
and signal transduction of UV-B irradiation in Arabidopsis thaliana.
2. Scherbak, Nikolai (2005). Characterization of stress-inducible shortchain dehydrogenases/reductases (SDR) in plants. Study of a novel
small protein family from Pisum sativum (pea).
3. Ristilä, Mikael (2006). Vitamin B6 as a potential antioxidant. A study
emanating from UV-B-stressed plants.
4. Musa, Klefah A. K. (2009). Computational studies of photodynamic
drugs, phototoxic reactions and drug design.
5. Larsson, Anders (2010). Androgen Receptors and Endocrine
Disrupting Substances.
6.
Erdtman, Edvin (2010). 5-Aminolevulinic acid and derivatives
thereof. Properties, lipid permeability and enzymatic reactions.
7.
Khalaf, Hazem (2010). Characterization and environmental
influences on inflammatory and physiological responses.
8.
Lindh, Ingrid (2011). Plant-produced STI vaccine antigens with
special emphasis on HIV-1 p24.
9.
El Marghani, Ahmed (2011). Regulatory aspects of innate immune
responses.
10. Karlsson, Mattias (2012). Modulation of cellular innate immune
responses by lactobacilli.