UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series no. 1236 ISSN: 0346-6612 ISBN: 978-91-7264-710-7 Editor: The Dean of the Faculty of Medicine

Immunopathogenesis of Relapsing Fever Borreliosis

Marie Andersson

Department of Molecular Biology Umeå University

Sweden 2009

Cover: AFM micrograph of Borrelia duttonii spirochetes

Copyright © 2008 by Marie Andersson Printed by Print & Media, Umeå, 2008

“Set a new course. There’s coffee in that nebula”, (Stardate: 49373,4), Captain Janeway, USS Voyager

Table of contents 

Abstract ................................................................................... 1

Papers in this thesis .......................................................... 2

Introduction .......................................................................... 3

1. History of relapsing fever .....................................................3

2. General characteristics of Borrelia species ................... 6

Morphology .................................................................................... 6

Genetic organization ....................................................................... 7

Surface proteins ............................................................................... 7

Motility ........................................................................................... 8

Cultivation of Borrelia ................................................................... 9

3. Classification of Borrelia ................................................... 10

4. Transmission and maintenance of Borrelia ................. 13

The tick vector ............................................................................... 13

The human body louse .................................................................. 15

5. The disease ............................................................................. 16

Epidemiology ................................................................................. 16

Clinical manifestations of the disease .......................................... 17

RF in pregnancy ............................................................................ 19

Diagnostics and Treatment .......................................................... 20

Prevention ..................................................................................... 21

Animal models .............................................................................. 22

6. The immune system during infection ............................... 24

Innate immunity .......................................................................... 24

Immediate effect: humoral mediators ......................................... 24

Secondary effect: cell-mediated innate immunity ....................... 27

Adaptive immunity ....................................................................... 28

Lymphocytes and their receptors ................................................ 28

Antibodies ..................................................................................... 29

The spleen ..................................................................................... 30

Brain immune response ................................................................ 31

Immune response during pregnancy ........................................... 32

7. Host-pathogen interactions during borreliosis .......... 34

Deposition in the skin .................................................................. 34

Penetration of tissue and barriers ................................................ 35

Interactions in the circulation ..................................................... 36

Antigenic variation ....................................................................... 38

Tissue tropism .............................................................................. 39

Clearance of a blood-borne pathogen .......................................... 39

Complement and serum resistance ............................................. 40

Antibody clearance ........................................................................ 41

Interactions with phagocytic cells ............................................... 42

Toll-like receptor signaling .......................................................... 44

The balance between pro- and anti-inflammatory mediators .... 46

Borrelia in the brain .................................................................... 48

Aims of the thesis ............................................................. 51

Results and Discussion ................................................. 52

PAPER I .............................................................................................52

In situ brain response in brain and kidney during early relapsing fever borreliosis ...............................................................

PAPER II ......................................................................................... 56

Persistent brain infection and disease reactivation in relapsing fever borreliosis ...............................................................

PAPER III ......................................................................................... 60

Pregnancy complications and trans-placental transmission of relapsing fever borreliosis ...........................................................

PAPER IV ......................................................................................... 65

Enhanced inflammatory response to relapsing fever during pregnancy .........................................................................................

Concluding remarks ....................................................... 72

Sammanfattning på svenska ...................................... 74

Acknowledgements ......................................................... 77

References ............................................................................ 79

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Abstract Relapsing fever (RF) is caused by different species of Borrelia transmitted by soft ticks or by the human body louse. Illness is characterized by reappearing peaks of high concentrations of spirochetes in blood, concordant with fever peaks separated by asymptomatic periods. Neuroborreliosis is one of the most severe manifestations of RF borreliosis. To understand the immune response during early RF, we analyzed immune cells in brain and kidney of mice infected with B. crocidurae during the acute infection. Our results indi-cate that brain defense is comprised primarily of innate immune cells. De-spite the infiltration of innate immune cells, Borrelia was not completely eradicated. A failure of the host brain to clear the bacteria may give the pa-thogen a niche where it can persist. Using our mouse model, we revealed that Borrelia duttonii could persist in the mouse brain for up to 270 days, with-out being present in the circulation. The infection was silent with no change in host gene expression, and the spirochetes could re-enter the circulation after immunosuppression. We propose that the brain is used by the pathogen to evade host immunity and serves as a possible natural reservoir for B. dut-tonii, a spirochete that has rarely been found in any mammalian host other than man. Borrelia-induced complications during pregnancy have been re-ported, and are especially common in RF. In our established mouse model of gestational RF, we could show that the fetuses suffered from severe patholo-gy and growth retardation, probably as a consequence of placental destruc-tion. We could also show trans-placental transmission of the bacteria leading to neonatal RF. Surprisingly, pregnant dams had a lower bacterial load and less severe disease, showing that pregnancy has a protective effect during RF. We have used the gestational RF model to investigate host factors favoring disease resolution. Because the spleen is the primary organ responsible for trapping and removing blood-borne pathogens, we have compared temporal changes in spleen immune cell populations and cytokine/chemokine induc-tion during the infection. Spleens of pregnant mice had earlier neutrophil infiltration, as well as faster and higher production of pro-inflammatory me-diators. This rapid, robust response suggests a more effective host defense. Thus, an enhanced pro-inflammatory response during pregnancy imparts a distinct advantage in controlling the severity of relapsing fever infection.

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Papers in this thesis  

I. Andersson, M., Nordstrand, A., Shamaei-Tousi, A., Jansson, A., Bergström, S. & Guo, B. P. (2007). In situ immune response in brain and kidney during early relapsing fever borreliosis. J Neuroimmunol 183, 26-32.

II. Larsson, C., Andersson, M., Pelkonen, J., Guo, B. P., Nordstrand, A. & Bergström, S. (2006). Persistent brain infection and disease reactivation in relapsing fever borreliosis. Microbes Infect 8, 2213-2219.

III. Larsson, C., Andersson, M., Guo, B. P., Nordstrand, A., Hagerstrand, I., Carlsson, S. & Bergström, S. (2006). Complications of pregnancy and transplacental transmission of relapsing-fever borreliosis. J Infect Dis 194, 1367-1374.

IV. Andersson, M., Larsson, C., Nilsson, I., Guo, B. P., & Bergström, S. (2008). Enhanced inflammatory response to relapsing fever during pregnancy. Submitted manuscript.

   

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Introduction 

1. History of relapsing fever

“In the first half of this century, relapsing fever was of consi-derable interest to microbiologists, not only because of the con-tinuing morbidity and mortality it was causing, but also be-cause the recognition by several early immunologists that the antigenic variation of relapsing fever was a useful model for studying the immune system” (Barbour & Hayes, 1986)

Relapsing fever (RF) has been recorded and described since an-cient times, even though the disease had not been given its con-temporary name and the causative agent had not yet been de-termined. An epidemic fever was described by Hippocrates, called the “ardent fever,” and during the following centuries preceding modern times, several outbreaks of epidemic fever had been suggested to be relapsing fever (Felsenfeld, 1971). In the middle of the 18th century, the first clinical features were well described in a documented outbreak in Ireland. This epi-demic spread over the British islands, and the disease was for the first time designated “relapsing fever” in 1843 (Felsenfeld, 1971; Southern & Sanford, 1969). From England, relapsing fever was introduced to the United States, causing outbreaks in popu-lations living under poor conditions. Most famous is the “Phila-delphia epidemic,” an outbreak that lasted for about 30 years in the eastern region. The epidemic also spread to the European continent, and Scandinavia was plagued by the epidemic during the Russian-Swedish war in 1788. The causative agent was not known, however epidemic outbreaks were commonly found in

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Figure 1. Ilya Ilyich Mechnikov (1845-1916). Russian immunologist and Nobel Prize Winner in Medi-cine in 1908. Attempted sui-cide by inoculating himself with B. duttonii.

poor and overpopulated areas. Eventually, by inoculating mon-keys with crushed lice from human relapsing fever patients, it was shown that the disease was spread by a vector: the human body louse Pediculus humanus humanus (Southern & Sanford, 1969). The organism causing the epidemic disease was discov-ered in 1868 by the German physician Obermeier, and named Spirocheta Obermeieri, but later on the organism was designat-ed Borrelia recurrentis (Felsenfeld, 1971). The biggest out-breaks of epidemic RF occurred during World War I and II when hundreds of thousands soldiers and civilians were in-fected.

A similar disease was described on the African continent for the first time by the famous explorer Dr. Livingstone in 1857, but the disease was already familiar to the inhabitants of the area. Dr. Livingstone called it “a human tick disease,” but it was not until the beginning of the 20th century that Cook noticed the presence of Borrelia in a blood sample of a patient in Uganda, a finding that was confirmed later by Ross and Milne, Dutton and Todd, and Koch. They demonstrated that Borrelia was the causative agent of the disease in humans and monkeys in East Africa, and that it was transmitted by the tick Or-nithodoros moubata. Early at-

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tempts to study the disease could be hazardous, as many re-searchers inoculated themselves with infected blood or the iso-lated organism. Dutton even died as a result of his trials (Fel-senfeld, 1971). Researchers were aware of the high mortality risk of the disease as demonstrated by the Russian immunologist and Nobel Prize Winner, Mechnikov, who tried to commit sui-cide by inoculating himself with RF Borrelia (Figure 1). Fortu-nately, he did not succeed, and his further work contributed greatly to the knowledge of phagocytosis (Nobel Lectures, 1967). Relapsing fever has since then been reported in almost all parts of the world, with some exceptions.

The adaptive capacity of the organism itself is intriguing in many ways. Early researchers have done extensive work in studying epidemiology, entomology, spirochete morphology and disease progression, topics that have been thoroughly reviewed by Southern and Sanford in 1969 and by Felsenfeldt in 1971. Immunologists have been attracted to the disease because of the phenomenon of antigenic variation, and Barbour and co-workers have been the leading researchers in understanding the molecular mechanism behind this phenomenon. Other contem-porary researchers have contributed to the gross understanding of the genome organization, metabolism, and host-pathogen in-teractions. Still, in the beginning of the 21th century, there are many unsolved issues, including the interactions between dif-ferent RF Borrelia species and their different hosts, mammals or arthropods. Thus, there are still many unsolved mysteries to be dealt with for future researchers interested in relapsing fever borreliosis.

2. General characteristics of Borrelia species

“In 1681, after examining material removed from the teeth of an old man, van Leeuwenhoek noted, ‘I found an unbelievably great company of living animalcules aswimming more nimbly than any I had ever seen up to this time. The biggest sort (whe-reof there were a great plenty) bent their body into curves in going forwards…’ It is probable that this large bent organism was a spirochete” (Holt, 1978).

Morphology

The causative agent of relapsing fever is a spirochete that be-longs to the genus Borrelia. Like all spirochetes, it has a long, helical/wave-shaped appearance with an axial fibril (Figure 2). The length varies between 3-25 µm and the diameter is between 0.2-0.5 µm (Felsenfeld, 1971; Holt, 1978). Different species are generally different sizes, but great diversity can also be seen within one infecting strain. This variation is a function of the nutritional conditions and the growth phase of the bacteria. Their morphology is further distinguished by a cell envelope composed of an outer sheath, a periplasmic space, and an inner membrane surrounding the cell cytoplasm. The outer sheath is not similar to other Gram-negative bacteria, as it is fluid (Bar-

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Figure 2. Transmission electron microscopy image of a Borrelia crocidurae spirochete. Photo courtesy of Christer Larsson.

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bour & Hayes, 1986) and does not contain any LPS (Takayama et al., 1987), but an extraordinarily high proportion of lipoprote-ins (Brandt et al., 1990). The outer sheath and the protoplasmic cylinder are separated by the periplasmic space, where the mo-tility apparatus, consisting of several periplasmic flagella, is si-tuated (Barbour & Hayes, 1986).

Genetic organization

Unique to Borrelia is their segmented genome composed of a linear chromosome, and a high content of plasmids, both circu-lar but also linear (Barbour & Garon, 1987; Barbour, 1988; Cas-jens et al., 2000; Hayes et al., 1988; Lescot et al., 2008; Plas-terk et al., 1985). The plasmids represent approximately one third of the genome and certain plasmids are essential for infec-tivity as many genes coding for virulence factors are situated on the plasmids (Barbour, 1993; Dai et al., 2006). Prolonged in vi-tro cultivation of B. burgdorferi results in loss of some of these plasmids (Barbour, 1988) and also loss of infectivity (Schwan et al., 1988), but RF Borrelia seems to remain infectious even af-ter several passages in vitro. Borrelia species have a relatively small genome compared to other bacteria (Fraser et al., 1997), and are missing many genes involved in metabolism.

Surface proteins

As mentioned above, Borrelia membranes are not similar to other Gram-negative bacteria as they have an unusually high content of lipoproteins. In RF species, the majority of lipoprote-ins belong to the variable major protein (Vmp) class. They are subdivided based on their molecular mass into variable small proteins (Vsp) with an apparent size of 19-22 kDa, and variable large proteins (Vlp) with a size of 35-40 kDa (Barbour et al.,

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1991). The lipoproteins give serotype specificity and are also highly immunogenic, a property that can be offset by antigenic variation, which will be discussed in more detail elsewhere. Borrelia species have a very limited metabolic and biosynthetic capacity of their own (Fraser et al., 1997), and are therefore de-pendent on uptake of nutrients from the surroundings. Porins are membrane-spanning channels that allow diffusion of nu-trients across the membrane. Isolation of outer membranes of Borrelia has revealed several porin activities of RF and Lyme disease (Shang et al., 1998; Skare et al., 1995) in lipid bilayer assays. Some porins have been identified in B. burgdorferi (Skare et al., 1996; Skare et al., 1997; Östberg et al., 2002) but the only one characterized so far in RF Borrelia is Oms38 (Thein et al., 2008).

Motility

All Borrelia species have a large degree of motility and move by way of a spinning/wave-like motion by rotating their flagellae clockwise or counter-clockwise (Goldstein et al., 1994). The fla-gellae are inserted at the ends of the bacterium and like the cy-toskeleton of an eukaryotic cell, they render the spirochetes wave-like in appearance (Wolgemuth et al., 2006). The number of flagellae differs between different species and can be as many as 30 (Charon et al., 1992). Mutants lacking flagellae are straight and also immobile (Sadziene et al., 1991; Sal et al., 2008). The role of motility in virulence has not been demon-strated in vivo, but it is possible that the movement may aid the spirochete in penetration and invasion of tissue as it has been demonstrated that a flagella mutant has reduced ability to pene-trate endothelial cell layers (Sadziene et al., 1991).

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Cultivation of Borrelia

Since the bacterium was discovered by Obermeier, there have been many attempts to cultivate the bacteria. It became clear that it had very stringent nutritional needs and was very diffi-cult to grow outside of its vector or reservoir hosts. Early at-tempts failed to cultivate Borrelia in standard laboratory me-dium; it could only be maintained in rat peritoneum and in em-bryonated eggs, or by passaging through mice (Felsenfeld, 1971). Eventually, a Borrelia medium formulation that was sup-plemented with serum, the amino sugar N-acetylglucosamine, a main constituent of peptidoglycan, and bovine serum albumin (BSA) was developed by Kelly. The formula was later improved by Barbour, Stoenner, and thereafter named the BSK medium. The serum and the BSA supply the medium with long-fatty ac-ids that Borrelia cannot synthesize on its own so supplementa-tion is required for successful maintenance. The addition of ge-latin to the medium increases the density of the culture, even though gelatin is not necessary for growth, as a viscous medium supports the motility of the bacteria, thus preventing aggrega-tion. Optimal growth is achieved within a temperature range of 30-37 °C and in a 5% CO2 incubator (Barbour, 1984). The gen-eration time for RF species in rodents is approximately six hours (Stoenner et al., 1982), but in culture they grow much slower.

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3. Classification of Borrelia

“In 1906 several groups of investigators studying the spiro-chete of American relapsing fever concluded it was different from those in Europe, Africa and Asia. Thus the name Spiro-chaeta novyi was given to the American strain” (Southern & Sanford, 1969).

Borrelia bacteria belong to the Spirochete phylum in which the human pathogenic bacteria Treponema and Leptospira also be-long. The species that are known to cause disease in humans are classified into Lyme disease or relapsing fever Borrelia. The Lyme disease group includes three genospecies that can cause disease in man: B. burgdorferi, B. afzelii, and B. garinii. The relapsing fever group is further divided into “new world” and “old world” Borrelia species based on the continent in which its vectors exist. Old world species are present on the European, African and Asian continents, whereas new world species are defined as those present in South and North America (Figure 3). There have been many different ways of classifying Borrelia. Many of the known relapsing fever species are classified accord-ing to their association with its arthropod vector. The ticks are adapted to live in their specific microhabitat, thereby making the Borrelia also specific for a certain geographic location (So-nenshine, 1997). The tick-Borrelia relationship is in many cases so specific that one Borrelia species cannot be transmitted by the vector specific for other Borrelia species (Barbour & Hayes, 1986). There has also been a division of species based on the range of mammals that they infect. For example, B. anserina can only produce infection in birds, whereas the louse-borne B. recurrentis is considered to be strictly human-specific. These methods are, however, not optimal for distinguishing all

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Borrelia species since some have a broader spectrum of hosts and can also be adapted to transmission from more than one tick (Ras et al., 1996). Modern techniques such as genotyping based on DNA sequences have lead to a revision of the phyloge-ny. To more accurately distinguish different species, the gene for 16S rRNA has been compared, but since a high degree of conservation makes the differences between sequences minute, it is hard to separate closely-related organisms (Fox et al., 1992; Ras et al., 1996). A commonly used technique for separation of subgroups within species is comparison of a non-coding inter-genic spacer (IGS), which is highly variable since it is not sub-ject to evolutionary pressure. A sequence situated between the 16S and 23S genes has been used for such analysis (Bunikis et al., 2004). The sensitivity of IGS analysis has been demonstrat-ed with the relationship between B. recurrentis and B. duttonii. Based on their vector relationships, they have traditionally been

Figure 3. Geographical distribution of some RF Borrelia species and their vectors (Adapted from Rebaudet & Parola, 2006)

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classified as a single species, but based on the high similarity of the IGS sequence from B. duttonii, it was suggested that they were more closely related (Scott, 2005). Actually, the sequenc-ing of several RF Borrelia genomes showed that B. recurrentis is a decaying strain of B. duttonii, with a truncated genome (Lescot et al., 2008). Another useful gene sequence that has been used to separate RF species from Lyme disease species is the glpQ gene (Bacon et al., 2004).

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4. Transmission and maintenance of Borrelia

“As it is metaphorically true for the chicken and the egg, it may never be known whether Borrelia were originally parasites of arthropods or of vertebrates” (Barbour & Hayes, 1986).

The tick vector

Relapsing fever is a zoonotic disease and, as with all Borrelia diseases, is transmitted by hematophagous arthropods, the vec-tor, to different vertebrates called the reservoir host (Barbour & Hayes, 1986). The bacteria are strictly dependent on the arth-ropod and the reservoir host to be maintained in nature, and have therefore a biphasic lifecycle where they alternate between infecting the vector and the vertebrate. The success of this al-ternating lifestyle is dependent on adaptation to different envi-ronments. Ticks transmitting Borrelia are divided into two ma-jor families: the hard-bodied tick (Ixodidae), and the soft-bodied tick (Argasidae). The hard-bodied ticks are vectors for Borrelia species causing Lyme disease, and also for Borrelia miyamoto, a species more similar to the RF group (Fukunaga et al., 1995). The largest group of the Argasidae family is the Or-nithodoros, which is the main vector for relapsing fever. As with all ticks, they are blood-suckers and they need the blood-meal for their development. Soft-bodied ticks live in close proximity to animals in burrows, caves, bird nests, pig-pens, or even in man-made habitats, and are often nocturnal feeders, coming out during the night to actively seek their host. The tick can sense the heat, CO2, and odors emanating from the animal, giv-ing the tick the signal to move towards the stimuli and attach itself to the host skin. Ticks are pool-feeders, so they tear and rip up a pool in the skin that becomes filled with body fluids and blood that they can engorge. Different families of ticks feed for

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different durations: Ixodes ticks are slow feeders and will stay on the host for several days before the feeding process is com-pleted. These ticks can in a single blood-meal engulf as much as 4-5 ml of blood. On the other hand, Ornithodoros ticks are fast feeders and can complete the blood-meal as fast as within mi-nutes, but usually within 1-2 hours. They do not consume as large volumes of blood as the Ixodiae tick; instead one tick can feed several times and on multiple hosts so the spreading fre-quency of the bacteria is higher (Sonenshine, 1997). During its lifecycle, a tick goes through three developmental stages: the larva, the nymph and the adult stage. All three developmental forms require a blood-meal before transformation to the next stage. The female Ornithodoros lays just a couple of hundred eggs, considered to be few in comparison to females of the Ixo-didae family that can lay as many as 23,000 eggs, but that is compensated by the ability of the Ornithodoros female to lay eggs several times during its adulthood. Some RF Borrelia can be transovarially transmitted so the larvae are infectious when they take their first blood-meal, further increasing the transmis-sion frequency. However, this occurs infrequently. Several fea-tures contribute to the success of ticks as vectors for pathogen transmission: a long life-span and durability, as they can resist starving for years, and an ability to infest a majority of verte-brates (Sonenshine, 1997).

The transmission of Borrelia occurs when a naïve larva, nymph or tick feeds on a Borrelia-infected animal. Borrelia replicates in the tick midgut and when their numbers increase, they mi-grate towards the tick internal organs such as the nervous tis-sue, the salivary glands and the coxal organs (Barbour & Hayes, 1986). The bacteria can persist in the tick vector for several years before they are transferred to a vertebrate host with the blood-meal (Sonenshine, 1997). When the infected tick feeds on

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a naïve animal, the bacteria are deposited into the vertebrate skin, rapidly migrating into the circulation and multiplying there, reaching high densities. The bacteria also leave the circu-lation and penetrate various tissues such as the liver, spleen, the kidneys, and the brain; at this time-point they may be undetect-able in the blood.

The human body louse

The human body louse, Pediculus humanus humanus, is the vector for Borrelia recurrentis, the cause of epidemic relapsing fever. Lice are relatively short-lived and molt three times before reaching the adult stage when they will stay alive for approx-imately 20 days. They are highly susceptible to cold and remain attached to the inner side of clothes, only moving to the skin when feeding. They feed typically five times per day (Raoult & Roux, 1999). Transmission of B. recurrentis to humans does not occur through the bite, instead the bacteria are transferred when the louse is crushed and the spirochete-containing hemo-lymph of the louse is rubbed into the bite site (Barbour & Hayes, 1986). After Borrelia is ingested by the louse, it takes the route from the midgut to the hemolymph where it multiplies. It dis-seminates to all cavities of the body, without entering the tis-sues (Southern & Sanford, 1969).

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5. The disease

“Symptoms of relapsing fever vary with the immunity of the host, the strain of Borrelia that is involved, the phase of the ep-idemic, and a number of other less known or unknown factors” (Felsenfeld, 1971).

Epidemiology

Cases of tick-borne relapsing fever can occur sporadically and locally, often as a result of humans intruding into tick nesting areas. In the Western U.S., the disease is seasonal, occurring more often during the summers, and quite often the disease is caught by campers and hikers exposing themselves to rodent-infested cabins and shelters (Schwan et al., 2003). Commonly, people in these areas are not exposed much to the tick since the ticks are mainly feeding on their natural animal hosts (Sonen-shine, 1997). The disease can also be endemic, as it is present in parts of Africa where, for example, the vector O. moubata is adapted to human habitats. There, the prevalence of the disease is high, but it does not give any severe symptoms as inhabitants are constantly exposed to the bacteria, and thus have antibodies protecting them from severe disease. Those at greatest risk for developing severe disease are children (Barclay, 1990). Fatality rates can be around 2-5%, but as high as over 20% for children under one year old (Southern & Sanford, 1969). However, RF is a common cause of fever in many rural areas, with prevalence about 10% (Nordstrand et al., 2007; Vial et al., 2006). In cer-tain areas of Tanzania, infection with B. duttonii is estimated to be the sixth highest cause of hospital admissions and the se-venth highest cause of mortality among children (Barclay, 1990).

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Also, people visiting the endemic areas who have not had any prior exposure to the bacteria are at great risk, as they have not developed any immunity to the bacteria (Southern & Sanford, 1969). The severity of the disease is variable, depending on what species that is the infecting agent. New world species are consi-dered to induce a less severe disease with fewer relapses than old world species. The most serious disease is caused by the louse-borne relapsing fever agent B. recurrentis, with fatality rates of up to 40% if untreated (Raoult & Roux, 1999; Southern & Sanford, 1969). Louse-borne RF is considered to be a separate disease from tick-borne RF even though the symptoms are simi-lar. Due to the nature of the vector, louse-borne RF is epidemic. Epidemic outbreaks occur when the lice are easily spread: under conditions of overcrowding and poor hygiene, such as in refugee camps. Since lice live in human clothing and are sensitive to cold weather, outbreaks are more prevalent during the winter (Raoult & Roux, 1999). Elimination of the body louse through better hygienic conditions has made outbreaks of the disease limited to a few foci.

Clinical manifestations of the disease

Borrelia can cause two different diseases: relapsing fever and Lyme disease. The latter is characterized by a localized skin in-fection, erythema migrans, at the site of the tick bite that is fol-lowed by a disseminated infection. Lyme disease Borrelia do not cause high bacteremia, instead a few bacteria spread to or-gans such as the brain, heart and the joints, and may cause per-sistent disease if untreated. Relapsing fever, on the other hand, is an acute disease with a high degree of bacteremia. The incu-bation period is 2-18 days after the tick bite, but in contrast to Lyme disease, RF does not cause erythema migrans, rendering the start of the disease difficult to detect. The onset is very ab-

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rupt as the infected patient develops a sudden high fever, ac-companied by chills, severe headache and other undefined flu-like symptoms. Organs that are involved during the early infec-tion are the spleen and the liver, with hepato/splenomegally be-ing a common symptom; however Borrelia spirochetes are highly invasive and can be found in a variety of other tissues. The symptomatic period lasts for 3-5 days. Thereafter the pa-tient experiences a pronounced temperature elevation, followed by the crisis, a sudden temperature drop down to normal or even lower temperatures than pre-infection. The febrile period

is a result of a massive bacteremia as the Borrelia multiply in the blood reaching as many as 108 bacteria per milliliter of blood (Figure 4). After the temperature has gone down to nor-mal, a period of well-being occurs when no bacteria are detected in the blood. The afebrile p sts for 4-7 days whereafter

Figure 4. Spirochetemia. High numbers of B. duttonii in the mouse circulation at day 5 post-infection.

eriod la

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the disease relapses with subsequent fever peaks, spirochete-mia, and the aforementioned symptoms. An average of three relapses are common, but as many as 13 have been recorded. The first attack is commonly more severe and prolonged than the following relapses. Usually, the louse-borne relapsing fever patients experience fewer relapses (Southern & Sanford, 1969). Thrombocytopenia and low blood cell counts causing anemia are other common features. These may be consequences of Bor-relia interactions with platelets and erythrocytes, so-called erythrocyte rosetting. One serious pathological consequence of rosetting can be microemboli, causing the blood flow to arrest and subsequent hemorrhaging in the tissue, as it has been dem-onstrated in a mouse model (Shamaei-Tousi et al., 1999). In-volvement of the central nervous system (CNS) is common, as the bacteria can penetrate the blood-brain barrier. Physical symptoms of brain infection may be meningitis, hemiplegia, fa-cial palsy and neuritis, but mental disorders also may occur (Cadavid & Barbour, 1998; Southern & Sanford, 1969).

RF in pregnancy

Tick-borne relapsing fever is associated with a high incidence of pregnancy complications, such as spontaneous abortions, pre-term delivery and low birth-weight of neonates and perinatal death (Barclay, 1990; Bryceson et al., 1970; Jongen, 1997; Southern & Sanford, 1969; van Holten et al., 1997). Several case studies report neonatal infection in infants whose mothers were ill during delivery. In most of the cases, it is impossible to rule out infection after birth. But even if rare, in a few cases trans-placental infection during maternal RF (Brasseur, 1985; Fuchs & Oyama, 1969) or Lyme disease (Schlesinger et al., 1985) infec-tion have been suggested, although Lyme disease had not been associated with increased risk for pregnancy complications

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(Strobino et al., 1993). Adverse pregnancy outcome has been observed in Lyme disease (Markowitz et al., 1986; Walsh et al., 2007) and also been demonstrated in animal models, but not in association with spirochetal invasion of fetal tissue (Silver et al., 1995; Weis et al., 1997). Trans-placental transmission of RF spi-rochetes has been demonstrated in animals (Larsson et al., 2006a; Walker et al., 2002). Some case studies indicate that pregnant women are more susceptible to RF, having higher spi-rochetemia and developing a more severe disease (Dupont, 1997; Jongen, 1997; Mayegga et al., 2005).

Diagnostics and Treatment

RF can be very difficult to diagnose as the first symptoms are diffuse and resemble influenza. RF can also be obscured by ma-laria infection (Barclay, 1990; Nordstrand et al., 2007), a dis-ease that is common in the same areas as African RF is preva-lent. Other factors contributing to the difficulty of diagnosing RF is the nocturnal and fast-feeding behavior of the tick and the lack of a skin manifestation which allows the tick bite to go un-noticed. It is possible to diagnose an ongoing Borrelia infection by examining a blood sample from the infected patient, as the bacteria are readily detected by dark-field microscopy, or by Wright-Giemsa staining. However, detection demands a suffi-cient number of bacteria in the blood, such as during the first peak of spirochetemia, a time period that is very easily missed. However, the sensibility of this method can be improved by re-moving the red blood cells and concentrating the spirochetes in the plasma (Larsson & Bergström, 2008). It is also useful to in-oculate susceptible laboratory animals with blood from sus-pected RF patients, and examine the blood of the animal for a developing spirochetemia. This is a highly sensitive assay since a single Borrelia bacterium is enough to successfully establish

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the infection to laboratory animals (Barbour & Hayes, 1986), albeit impractical as a diagnostic method.

Serological tests have been used, but have not been highly sensi-tive due to variation in the Vmp’s and cross-reactivity with other Borrelia species, a major draw-back in areas where both RF and Lyme disease species are prevalent. GlpQ, a protein that is con-served among all members of RF Borrelia, but is absent in Lyme disease spirochetes, can be used to discriminate between these two agents (Nordstrand et al., 2007; Schwan et al., 1996). Although it is possible to diagnose RF by serology, treatment may be too late. The antibody response towards the bacteria de-velops after approximately 4-6 days (Yokota et al., 1997), a time-point when the bacteria already have disseminated to tis-sues such as the brain (Andersson et al., 2007; Cadavid & Bar-bour, 1998; Nordstrand et al., 2001), where antibiotic penetra-tion is limited.

Borrelia species are susceptible to antibiotic treatment, and treatment is in most cases curative (Bryceson et al., 1970; So-nenshine, 1997). Commonly used antibiotics are penicillin, erythromycin and tetracyclines. One risk of antibiotic treatment is the occurrence of a Jarish-Herxheimer reaction, a condition that resembles sepsis where the temperature suddenly rises fol-lowed by a drop in blood pressure, with risk of shock and cardi-ovascular collapse. The reaction can be life-threatening and is provoked by release of bacterial components when bacteria are lysed by the antibiotic (Bryceson, 1976).

Prevention

Since there is no current vaccine against RF, the best way to protect humans from the infection is to avoid or eliminate the

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vectors that transmit the disease. This can be difficult in areas where the vector lives in close association with humans, as evi-dent for Ornitodoros moubata. These ticks live in cracks and crevices in the mud-huts, a common type of house in sub-Saharan Africa (Sonenshine, 1997). Over 80% of traditional mud dwellings are infested with O. moubata (Barclay, 1990), making the inhabitants constantly exposed to the tick. Im-provement of the houses is a method for prevention but insect repellants and bed-nets have also been used and proven to be successful (Sonenshine, 1997).

The only strategy to eliminate louse-borne RF is eradication of the louse, but since louse infestation is a symptom of poverty, eradication can only be successful by improving the hygienic standard.

Animal models

Experimental infection of animals can mimic the disease of hu-mans in RF research. Animals such as monkeys, mice, ham-sters, rabbits and guinea pigs have been widely used (Southern & Sanford, 1969). The most common animal model used is the mouse, Mus musculus. Its genome is sequenced and there are a great number of genetically modified strains available. Mice display the disease in a similar way as humans, with high spiro-chetemia and invasion of various organs (Andersson et al., 2007; Nordstrand et al., 2001; Shamaei-Tousi et al., 2001). The high morbidity and mortality rates of B. duttonii infection have been demonstrated in various mouse models. Prior to death, the animals are in very poor condition, with ruffled fur, signs of de-hydration, sunken eyes and pale ears and paws accompanied by low platelet and red blood cell counts and anemia. Mice also show spleen and liver enlargement (Wright & Woodrow, 1980),

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pointing to the involvement of the reticuloendothelial system in the disease process. Interestingly, sensitivity to Borrelia infec-tion can vary between different inbred mouse strains, as dem-onstrated by the higher susceptibility of the C3H/He strain compared to the C57BL/6 strain, which is considered to be re-sistant. In the Lyme arthritis mouse model, C3H/He mice have more severe arthritis than the C57BL/6 mice, even though they have similarly high bacterial load in the tissue (Ma et al., 1998; Wooten & Weis, 2001). In RF, we have also observed that B. duttonii infections in C3H/HeN mice cause more severe disease progression as well as higher bacteremia (author’s observation), indicating that the genetic composition of the animal deter-mines the susceptibility to RF borreliosis.

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6. The immune system during infection

“Whenever the organism enjoys immunity, the introduction of infectious microbes is followed by the accumulation of mobile cells, of white corpuscles of the blood in particular which ab-sorb the microbes and destroy them. The white corpuscles and the other cells capable of doing this have been designated "phagocytes", i.e. devouring cells, and the whole function that ensures immunity has been given the name of ‘phagocytosis’” Ilya Mechnikov, (Nobel Lectures, 1967).

Innate immunity

Innate immunity is the initial defense that can directly and without prior encounter eliminate infectious agents (Figure 5). The response is unspecific and recognizes “non-self” molecules. It is composed of the physical layers of the body, such as the skin and the mucosal layers of the nasal-pharyngeal tract, but also by components that are constitutively present. In addition, cells that can remove foreign components without prior activa-tion are important. These cells have the capability to act direct-ly, and therefore are the first immune mediators that an invad-ing microbe will encounter. The primary effect includes pre-formed components and is available immediately or within an hour of infection, whereas the secondary effect, composed of cells, is set into motion within four hours (Janeway, 2005).

Immediate effect: humoral mediators

The “inflammatory response” is the general term for a process that is initiated upon injury or infection. It involves changes in the vascular tissues and leads to accumulation of plasma pro-

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teins and innate immune cells that act to aid the body to ob-struct the infection.

The acute phase response is composed of a battery of proteins produced mainly by the liver that have roles in regulating the inflammatory response but also directly killing or marking pa-thogens for destruction. Plasma concentrations of acute phase proteins can decrease as well as increase in response to infec-tion (Gabay & Kushner, 1999).

The complement system is a set of pre-made plasma proteins that can attack pathogens in the circulation, spontaneously upon encounter, or by the aid of an antibody bound to a patho-gen surface. Commonly, these proteins are inactive or re-strained by complement regulatory proteins until pathogen en-counter, when a proteolytic cascade is initiated that ends with the formation of a C3 convertase. This convertase is the main component responsible for generation of the effector proteins C3b, C3a, C5a, and the components of the membrane attack complex (MAC). C3b binds to pathogen surfaces and is in turn recognized by complement receptors on phagocytic cells. This process is called opsonization and increases these cells’ ability to phagocytose. C3a and C5a act as chemoatttractants for im-mune cells and the MAC-complex will insert into the pathogen membrane, leading to lysis of the microbe. The system can be activated in three ways: the classical pathway which is initiated by the formation of an antigen-antibody complex, the alterna-tive pathway, with spontaneous cleavage of component C3 and deposition of C3b on the pathogen surface, and finally, the mannan-binding lectin pathway. The mannan-binding lectin pathway is initiated by the binding of lectin to polysaccharides on a pathogen surface. As the latter two are activated indepen-dently of antibodies, they can act early. All three pathways di-

Figure 5. Overview of the immune system. Both the innate and the adaptive immune responses contain cellular as well as humoral media-tors. The link between the systems is antigen-presenting cells. Upon an-tigen presentation to a T-cell, it can “help” the B-cell to be a antibody secreting cell, the plasma cell.

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verge at the formation of the C3 convertase, (Janeway, 2005; Walport, 2001).

Natural antibodies are pre-made, circulating IgM present in low titers. These immunoglobulins can neutralize pathogens directly or indirectly, by activation of the classical pathway of the com-plement system, or by targeting the pathogens to lymphoid or-gans such as the spleen. IgM molecules have a low affinity to many repetitive molecules, such as carbohydrates, on pathogen surfaces. The major source of natural antibodies is the non-

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conventional B1b-cell which is found in the peritoneal cavity (Ochsenbein & Zinkernagel, 2000).

Secondary effect: cell-mediated innate immunity

The secondary effects on the innate immune response are trig-gered by the recognition of pathogens via germ-line encoded pattern-recognition receptors (PRR) expressed on innate im-mune cells. These receptors have a broad specificity and recog-nize conserved repetitive microbial motifs such as LPS, man-nose, and peptidoglycan. Molecules containing these motifs are abundantly present on many microorganisms. Examples of these receptors are Toll-like receptors (Lien et al., 1999) and the mannose receptor (East & Isacke, 2002). Other receptors rec-ognize opsonized microbes, such as the Fc-receptors that bind to antibodies on pathogen surfaces (Fridman, 1991) and comple-ment receptors that recognize products from the complement cascade deposited on microbial surfaces. Phagocytic cells ex-press many PRR (Janeway, 2005).

The main phagocytic cells are neutrophils and macrophages. Neutrophils are present in the blood in large numbers and can rapidly be recruited to an infection site. These cells non-selectively clear away foreign matter, but have also the ability to kill pathogens extracellularly by secreting chemical compounds. Macrophages and dendritic cells (DC) are more potent phago-cytes that additionally have antigen-presenting capacity. Upon binding of the ligand and subsequent phagocytosis, they become activated to secrete cytokines, and to more effectively remove pathogens through phagocytosis. Finally, they can migrate to secondary lymphoid tissues and present the foreign antigen to B- and T-cells (Figure 5), thereby initiating the adaptive im-mune response (Dale et al., 2008; Janeway, 2005).

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Cytokines are immune response mediators that affect the beha-vior of immune cells. They regulate the intensity of the immune response as they can have pro-inflammatory actions (enhancing the immune response), as well as anti-inflammatory functions (down-regulating the immune response). Chemokines are che-moattractant molecules that stimulate migration of immune cells to a site of infection (Janeway, 2005).

Adaptive immunity

The adaptive immune response is composed of highly specia-lized immune cells, the lymphocytes, that can recognize and eliminate pathogens efficiently and with high specificity. These cells have the ability to recognize and remember specific patho-gens, and to mount stronger attacks each time the pathogen is encountered, so-called immunological memory. As lymphocytes are activated through innate immunity (Figure 5), their re-cruitment is delayed on the first encounter so they do not ap-pear until after about four days, but since immunological mem-ory is established, the response will be immediate upon second-ary challenge.

Lymphocytes and their receptors

Lymphocytes include the T-cell and the B-cell. As immature cells, they migrate to lymphoid organs, the T-cells to the thymus and the B-cells to the spleen or to the lymph nodes. At these sites they mature through different stages to recognize specific foreign antigens and ignore “self-molecules,” so that only those that pass the selection survive and are allowed to mature. When they are mature, they express specific antigen receptors, the T-cell receptor and the immunoglobulin receptor of the B-cell. It is these receptors that make the system adaptable. All receptors

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are generated by a series of gene rearrangements, a mechanism that generates a large number of different antigen receptors. Specific receptors are then uniquely expressed on each individ-ual lymphocyte. Not all lymphocytes with different variations will be involved in the clearance of an infection, as it is only those that encounter a matching antigen that will be active. En-counter of antigen leads to clonal expansion of that specific cell, so that all progeny cells express the same receptor (Janeway, 2005). Depending on the properties of the infectious agent, the adaptive response can be cell-mediated or antibody-mediated. Intracellular pathogens provoke a cell-mediated response where cytotoxic T-cells directly kill the infected cell. Extracellular pa-thogens provoke an antibody-mediated response where B-cells produce large amounts of specific antibodies that can neutralize the pathogen. Common for both mechanisms is the generation of long-lived memory cells (Figure 5) that can react immediately without activation if the same infectious agent is encountered (Janeway, 2005).

Antibodies

Antibodies are the secreted form of the B-cell receptor that can diffuse into the circulation and tissue to act distantly from the producing cell. They are composed of two heavy chains and two light chains that are paired together to build a Y-shaped mole-cule where the two arms bind to the antigen and the heavy chain C-region, the Fc-part. The Fc-stem is recognized and bound by immune cells with Fc receptors. Antibodies are divided into classes, so-called isotypes, determined by which Fc domain they are displaying, giving the isotypes different effector functions. At the beginning of the B-cell developmental process, all anti-bodies are of the IgM isotype, but upon maturation they will switch their heavy chain C-region and start to express their

functional Fc stem, and produce IgG, IgA, or IgE antibodies. The switch is directed by cytokines but also by activation signals from T-cells (Janeway, 2005).

The spleen

The spleen is the largest secondary lymphoid organ in the body, and it is specialized in filtering the blood. It is therefore in-volved in immune responses to blood-borne pathogens. A spleen is composed of two different functional compartments (Figure 6). The red pulp is specialized for collection of blood-borne debris and bacteria, but it also removes damaged and se-nescent red blood cells. Macrophages are the dominant immune cell, and responsible for such removal. White pulp is the lym-phoid compartment where T- and B-cells reside. Within this

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Figure 6. Morphology of a mouse spleen. Immunohisto-chemistry staining of an uninfected mouse spleen. Dark stained areas represents macrophages in the red pulp posi-tive for the F4/80 marker. WP=white pulp, RP=red pulp, MZ=marginal zone.

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compartment, T-cells can interact with antigen-presenting cells and passing B-cells, and it is also a site for clonal expansion and antibody production by B-cells (Cesta, 2006; Janeway, 2005). At the interface of the white and the red pulp is the marginal zone (MZ). In this area, blood-flow is slowed and blood-borne particles are efficiently trapped by resident macrophages, the MZ macrophages (Aichele et al., 2003). The MZ also contains MZ B-cells which are involved in T-cell independent immune responses (Balazs et al., 2002), making it to an area for antigen screening and processing, but also for initiation of immune res-ponses (Kraal, 1992; Mebius & Kraal, 2005). Most of the blood flows through the spleen, making it a superior organ for screen-ing the blood for blood-borne pathogens.

Brain immune response

The central nervous system (CNS) is the most sensitive system in the body, and it is essential to restricting trafficking of poten-tially harmful molecules and cells from the blood into the CNS. The interface of the blood and the CNS is protected by the blood-brain barrier (BBB) and the blood-cerebrospinal barrier (BCB). These barriers are composed of microvascular endo-thelial cells adjoined by tight junctions that restrict paracellular diffusion. A specific transport system allows only essential sub-stances to pass the barrier to support normal neuronal function (Hawkins & Davis, 2005). The passage of leukocytes is strictly regulated; paracellular migration is a multi-step process involv-ing both endothelial cells and leukocytes, leading to modifica-tions and disruption of the tight junctions (Persidsky et al., 2006). Immune cells from the circulation can therefore not re-spond to a brain infection as rapidly as they can respond in oth-er tissues. Although considered to be an immune-privileged or-gan, the brain is not defenseless and can mount an immune re-

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sponse through the microglia cells, a macrophage-like immune cell. These are resident brain cells that remain inactive until stimulated by an infection or an injury, whereupon they rapidly and efficiently remove the infectious agent. Microglia express many TLR’s and have the same functions as the macrophages outside of the CNS (Olson & Miller, 2004). Antibodies cannot pass the BBB, but B-cells can migrate in small numbers into the brain and produce antibodies intrathecally (Anthony et al., 2003; Meinl et al., 2006). It has also been demonstrated that cells of the CNS express a variety of chemokines and cytokines that can modulate the BBB integrity, and activate resident cells and infiltrating immune cells (Gelderblom et al., 2007a; Kie-lian, 2004).

Immune response during pregnancy

Pregnancy is associated with a change in the immune-competence of the mother in order to protect the fetus from re-jection, as it is partly non-self due to paternal antigens. As a re-sult of progesterone production, the cytokine profile is modified towards a Th2 cytokine profile with secretion of anti-inflammatory cytokines. This leads to suppressed cell-mediated immunity and a dominating humoral response (Druckmann & Druckmann, 2005). The shift in competence can leave pregnant women more susceptible to infections with intracellular patho-gens, such as Plasmodium, Salmonella and Listeria (Abram et al., 2003; Okoko et al., 2003; Pejcic-Karapetrovic et al., 2007), and also lead to more severe infection with these pathogens. But generally, pregnant women are not considered to be more sus-ceptible to extracellular pathogens. Suppression of the specific, acquired immune response is instead compensated for by acti-vation of the non-specific immune response, with increased plasma levels of acute phase proteins as well more monocytes

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and granulocytes with an activated phenotype (Sacks et al., 1999; Sacks et al., 2003). The down-regulation of cell-mediated immunity can also have beneficial effects on chronic inflamma-tory diseases such as arthritis (de Man et al., 2008; Draca, 2002; Moro et al., 2001).

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7. Host-pathogen interactions during borreliosis

“On this hypothesis, disease would be a fight between the mor-bid agent, the microbe from outside, and the mobile cells of the organism itself. Cure would come from the victory of the cells and immunity would be the sign of their acting sufficiently to prevent the microbial onslaught”, Ilya Mechnikov, (Nobel Lec-tures, 1967).

During RF infection, the bacteria occupy a variety of tissues constituting different environments: the skin, the circulation, and eventually peripheral organs with their own specific milieu. The spirochetes will also encounter several components of the host immune system. Some of these components can be detri-mental for the bacteria, but there are also components that can aid the spirochete in invasion of tissues.

Deposition in the skin

The infection starts with deposition of the spirochete into the skin during the tick or louse blood-meal. A tick bite is painless and goes often unnoticed due to chemicals that are secreted along with the tick saliva. Tick saliva contains pain killers, anti-coagulatory and anti-inflammatory substances that aid the tick in obtaining a successful blood-meal (Ribeiro et al., 1985; So-nenshine, 1997; Waxman et al., 1990). In Ixodes ticks, the vec-tors for B. burgdorferi, B- and T-cell inhibitory proteins have been characterized (Garg et al., 2006; Gillespie et al., 2001; Hannier et al., 2004) as well as salivary anti-complement activi-ty (Ribeiro et al., 1987). Tick saliva also inhibits the activity of polymorphonuuclear (PMN) leukocytes, reducing the killing of the spirochete (Montgomery et al., 2004). As the tick is able to take a blood-meal unnoticed, this is also advantageous for the

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spirochete as they may more easily gain access to the blood-stream unnoticed.

Penetration of tissue and barriers

RF Borrelia penetrates both extracellular matrix (ECM) and the endothelial lining of the blood vessels. Their typical shape and movement aid in penetration of the tissue, demonstrated by the lost ability of flagella mutants to penetrate endothelial layers (Sadziene et al., 1991). Penetration through the microvascular endothelial cell layers and ECM by the use of a host protease system is well documented. Borrelia species have no endogen-ous protease activity (Klempner et al., 1996), but they bind components of the plasminogen activation system (PAS), both the inactive zymogen plasminogen and its activator, creating plasmin activity at the polar ends of the spirochete (Klempner et al., 1996). Plasmin is a broad-spectrum serine protease that de-grades fibrin clots and components of the ECM as fibronectin (Gebbia et al., 1999). The importance of PAS has been demon-strated both in vitro and in vivo as plasmin-coated spirochetes can degrade several substrates (Coleman & Benach, 2000) and also more efficiently penetrate endothelial cell layers (Coleman et al., 1995). Studies in plasminogen-deficient mice (plg-/-) have demonstrated that spread of RF Borrelia to tissues is delayed when the PAS is missing, and there is also a lowered bacterial load in the brain and the heart of infected animals (Gebbia et al., 1999; Nordstrand et al., 2001). As a secondary effect, the proteolytic effect of plasmin can further activate matrix metal-loproteinases (MMP’s) (Gebbia et al., 2001), creating a broader spectrum of substrates that can be degraded. The use of host proteases clearly facilitates spreading of the spirochete even though the activity is not a critical factor for initially reaching

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the circulation since plg -/- mice also develop spirochetemia similar to wild-type mice (Gebbia et al., 1999).

Interactions in the circulation

Some old world RF Borrelia species, mainly B. duttonii, B. cro-cidurae, and B. hispanica, frequently aggregate red blood cells, a phenomenon called erythrocyte rosetting (Figure 7). In vitro models have suggested that it is a mechanism of immune eva-sion. It is possible that rosetting increases the threshold for immune response initiation, as B. crocidurae which readily ro-settes erythrocytes, has a longer duration of the initial spiroche-temia than the non-rosetting strain B. hermsii. Additionally, the rosetting strain do also mount a delayed antibody response in comparison (Burman et al., 1998). Another hypothesis con-cerning the erythrocyte interaction is that the spirochete is graz-ing and picking up nutrients from the cells. Usually, rosetting is only observed when the spirochetes reach a certain density in the blood, such as during the first peak of spirochetemia (au-thor’s observation). In the murine model of B. duttonii infec-tion, we have calculated as many as one billion bacteria in every milliliter of blood (Larsson et al., 2006a), and obviously a my-riad of nutrients are needed to maintain the growth of the popu-lation. One piece of evidence pointing towards the grazing theory was demonstrated by Petterson and co-workers. When compared to LD Borrelia species, which never reach high densi-ties in the blood, RF Borrelia species exclusively have genes that enable them to use purines from serum as metabolites for syn-thesis of RNA and DNA. The purine hypoxanthine is very abun-dant in human plasma and produced by red blood cells, so it is likely that the interaction with erythrocytes is a mechanism which provides with this and other metabolites that are needed for growth (Pettersson et al., 2007). As anemia and low red

blood cell counts are common features of RF, it is possible that spirochete rosetting is damaging the cells and causing them to be prematurely removed. However, to date this hypothesis has not been tested. On the other hand, thrombocytopenia, also a common consequence of RF, is evidently caused by spirochete-

platelet interactions. B. hermsii attachment to platelets results in increased platelet loss and prolonged bleeding and the severi-ty of the thrombocytopenia is also correlated with the degree of spirochetemia (Alugupalli et al., 2003b). In conclusion, the in-teraction of Borrelia with cells in the circulation is probably a strategy to increase and prolong the time the pathogen can be maintained within the host. Red blood cells can provide nu-trients or shelter from the host immune response, and the loss of platelets will augment penetration into distant tissues.

Figure 7. Erythrocyte rosetting. B. duttonii aggregating mouse red blood cells.

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Antigenic variation

Early researchers noticed that every relapse of the disease was caused by a new variant of the original infecting strain. Even-tually, it was evident that the relapsing feature was a result of antigenic variation of the dominant surface protein, the Vmp’s (Barbour et al., 1982; Stoenner et al., 1982). During a fever peak, the majority of the spirochetes express one type of Vmp, eliciting the host to mount an antibody response towards this serotype, subsequently clearing the organisms from the circula-tion. However, a second wave of bacteria, expressing a different Vmp, will multiply and cause the disease to relapse. These bac-teria will not be recognized by the immune response, thus evad-ing antibody-mediated killing. The relapses and subsequent clearance are repeated several times until the repertoire of dif-ferent variations is exhausted. Only one Vmp is expressed at a time, in a single expression locus, other variants are silent and located on several linear plasmids. Expression switch to a new Vmp occurs spontaneously through recombination, and arises with a frequency of approximately 10-4-10-3 per cell generation (Hayes et al., 1988; Meier et al., 1985; Plasterk et al., 1985; Stoenner et al., 1982). The genome of B. hermsii contains at least 59 silent Vmp cassettes and the single expression locus is situated on a 28-kb linear plasmid (Dai et al., 2006; Kitten & Barbour, 1990). All possible Vmp’s are never expressed at the same time within a population; instead the order of appearance of the serotypes follows a certain hierarchy. Some serotypes are more likely to be expressed in the first peak, whereas other sero-types are more frequently expressed at later peaks (Barbour et al., 2006; Dai et al., 2006). Antigenic variation contributes to maintenance of Borrelia in nature since it prolongs bacterial survival in the circulation, thereby increasing the chance of suc-cessful transmission to a naïve host (Barbour & Hayes, 1986).

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Tissue tropism

Additional functions of antigenic variation of the Vmp’s are not fully understood, but it has become evident that they also are involved in adaptation to various surroundings and determi-nants of tissue tropism. B. turicatae serotype Bt2 (VmpB) is the more common serotype in blood, reaching very high densities, whereas serotype Bt1 (VmpA) is more likely to be found in the CNS. Bt2 also causes more severe arthritis than Bt1, due to a tropism for joint tissue (Cadavid et al., 1994; Cadavid et al., 2001; Pennington et al., 1997). Expression of different surface proteins can also be a determinant for successful transmission from the vector to the mammalian host. RF B. hermsii expresses a specific Vmp in the tick, and this Vmp is replaced by another upon tick feeding, indicating that the dominant Vmp expressed in the tick is not needed for infection of the mammalian host. This conversion can also be induced in culture by changing the temperature (Schwan & Hinnebusch, 1998). Also, the Lyme dis-ease agent B. burgdorferi has a differential expression pattern dependent on the environment. The dominant surface protein OspA is expressed in the tick midgut, however, when the tick starts to feed on a warm-blooded animal, it is down-regulated and replaced with expression of OspC (Schwan et al., 1995).

Clearance of a blood-borne pathogen

A hallmark of RF is the high level bacteremia that occurs at the first peak, the crisis. Despite the high numbers of circulating bacteria at the crisis, they are almost completely undetectable the day after, showing that the clearance is very rapid (Alugu-palli et al., 2001; Arimitsu & Akama, 1973; Burman et al., 1998; Shamaei-Tousi et al., 1999).

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Complement and serum resistance

The complement system is one of the first humoral components that Borrelia will encounter when it enters the bloodstream. Several species of Borrelia activate the human complement sys-tem, but not all are killed by the complement as they are serum-resistant (McDowell et al., 2003; Meri et al., 2006; van Dam et al., 1997). The complement system is strictly regulated by host complement regulators that protect “self” cells from deposition of complement proteins on their surfaces. Serum-resistant Bor-relia species exploit these regulators to do the same. One me-chanism is recruitment of the complement inhibitor Factor H and Factor H Like Protein 1 (FHL-1). Binding of these regula-tors leads to degradation of C3b, preventing opsonization (Kraiczy & Wurzner, 2006; McDowell et al., 2003; Meri et al., 2006). This is, however, not the only way for the spirochete to interfere with the complement system; the resistance can also occur through inactivation of the MAC complex. In this case, Borrelia expresses a surface protein that mimics a human com-plement regulatory protein (Pausa et al., 2003). The importance of complement insensitivity in natural infection is, however, not clear as B. burgdorferi is still able to infect mice deficient in Factor H, and mice deficient in component C5 still are able to control the infection (Bockenstedt et al., 1993; Woodman et al., 2007). This discrepancy has also been demonstrated for RF Borrelia infection (Connolly & Benach, 2001). However, it is possible that resistance to complement killing will provide the spirochete with additional time in the circulation to escape to distant sites where antibodies are less accessible, increasing the survival of the bacteria.

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Antibody clearance

Infection of mice that cannot produce antibodies, such as SCID mice (Pennington et al., 1997) and B-cell deficient mice (Con-nolly & Benach, 2001) results in persistent spirochetemia, de-monstrating the importance of antibodies. The protective capac-ity of antibodies has been well documented. Arimitsu and Aka-ma could show that B. duttonii immune sera sampled after day 4 post-infection gave protection to challenged mice, whereas immune sera from earlier time-points did not (Arimitsu & Akama, 1973). In experimental B. duttonii infection, it was demonstrated that IgM antibodies were the first to appear after 4-6 days, followed by the different classes of IgG after 7-8 days, but only IgM and IgG3 conferred protection after passive im-munization (Yokota et al., 1997). IgG3 has also been demon-strated to be transferred to the offspring of through the milk (Morshed et al., 1993). There are different mechanisms by which antibodies can protect the host from infection: by activa-tion of the classical complement system, or by a direct bacteri-cidal effect as demonstrated for killing of B. burgdorferi (Sad-ziene et al., 1994). As the complement system does not seem to be a prerequisite for antibody-mediated protection (Connolly & Benach, 2001), it is likely that the latter mechanism is of greater importance. In a study by Barbour and Bundoc, protection by an IgM antibody specific for B. hermsii serotype Vlp7 was dem-onstrated, confirming that T-cell help and isotype switching is not needed for bacterial clearance (Barbour & Bundoc, 2001). Alugupalli and co-workers further investigated the IgM re-sponse and demonstrated that conventional B-cells were not required for successful clearance of the bacteria; instead it was associated with an expansion of the non-conventional B-cell subset, B1b cells (Alugupalli et al., 2003a). These cells are known to produce large amounts of IgM (Fagarasan & Honjo,

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2000). A specific IgM antibody towards a relapsing fever spiro-chete was found to be bactericidal in the absence of comple-ment, and under the microscope it was obvious that the antibo-dy disrupted the membrane of the bacteria (Connolly et al., 2004; LaRocca et al., 2008). Clearly, the IgM response is in-volved in the early control of the infection, but it may also be involved in long-term immune response; it was demonstrated that IgM were produced by B1b-lymphocytes capable of immu-nological memory (Alugupalli et al., 2004). IgM can also be produced by marginal zone (MZ) B-cells (Fagarasan & Honjo, 2000). MZ B-cells contribute to elimination of the Borrelia since splenectomized mice have a defect in the ability to resolve the spirochetemia during high spirochetemia (Alugupalli et al., 2003a). Furthermore, Belperron et al., demonstrated that dep-letion of these cells during Borrelia infection results in an im-paired IgM response and enhanced pathogen burden (Belperron et al., 2005; Belperron et al., 2007).

Interactions with phagocytic cells

Both LD and RF Borrelia infections are characterized by disse-mination of the bacteria, pointing towards difficulties of the host in containing the initial infection. Neutrophils are not con-sidered to be the most efficient phagocyte of unopsonized Bor-relia (Montgomery et al., 2002; Spagnuolo et al., 1982; Suho-nen et al., 2000). On the other hand, neutrophil components can readily kill B. burgdorferi extracellularly (Lusitani et al., 2002; Lusitani et al., 2003), however the efficiency of the killing increases upon opsonization (Lusitani et al., 2002; Suhonen et al., 2000). B. burgdorferi-infected mice could control the infec-tion better if the neutrophil recruitment increased during early infection (Xu et al., 2007), indicating that an inefficient neutro-phil response may allow the bacteria to establish a disseminated

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infection. It has been demonstrated in vitro that B. crocidurae activates endothelial cells, promoting trans-endothelial migra-tion of neutrophils, a prerequisite for the neutrophil reaching the area of infection (Shamaei-Tousi et al., 2000). Still, RF Bor-relia can reach high densities in the blood and be found in dis-tant tissues within a few days, indicating some resistance me-chanism. A serine protease, BhpA, that renders increased resis-tance to neutrophil killing has been identified in RF species. This protease is absent in LD species (Guyard et al., 2006), which never grow to as high densities as RF species in the circu-lation, so the ability to resist neutrophil killing may be an adap-tation of the blood-borne spirochete. Still, having a rapid neu-trophil response may aid the host in containing the initial infec-tion and preventing bacteria from reaching too high numbers.

Macrophages can phagocytose and kill spirochetes without de-lay. The uptake can occur through the Fc-receptor on opsonized bacteria, but also independently of opsonization (Montgomery et al., 1993; Montgomery et al., 1994; Montgomery et al., 2002). Involvement of monocytes/macrophages is likely to be a prima-ry host strategy against Borrelia infection. Sambrii et al. showed that the uptake of RF Borrelia in the liver Kupffer cells was rap-id, pointing to the role of macrophages in the reticuloendotheli-al system in the killing of blood-borne pathogens (Sambri et al., 1996). During Lyme carditis, macrophages are the dominating immune cells involved in clearance; an inability to recruit ma-crophages leads to impaired control of bacterial levels (Mont-gomery et al., 2007). During acute RF, macrophage/microglia cells are also the dominant cells involved in brain infection with B. crocidurae (Andersson et al., 2007). Furthermore, it has been demonstrated that MCP-1, a main monocyte/macrophage chemoattractant is upregulated and produced as a response to LD spirochetes (Sprenger et al., 1997; Wang et al., 2008). The

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value of macrophages goes beyond phagocytosis as they are in-volved in orchestrating further actions of the immune response. As Borrelia are potent inducers of cytokine and chemokine pro-duction by monocytes/macrophages upon stimulation or pha-gocytosis (Cruz et al., 2008; Lazarus et al., 2008; Moore et al., 2007; Sprenger et al., 1997; Wang et al., 2008), macrophages may be a key mediator of the immune response to Borrelia.

DC’s are central to initiation of the adaptive immune response as they have the ability to activate naïve T-cells (Banchereau & Steinman, 1998), and these cells also readily phagocytose and digest Borrelia (Filgueira et al., 1996; Suhonen et al., 2003). However, the importance of DC’s in RF has not been evaluated. Their role as T-cell activators seems to be of minor importance as T-cells clearly are not needed for successful resolution of RF (Alugupalli et al., 2003a).

Toll-like receptor signaling

Toll-like receptors (TLR) are expressed on innate immune cells and recognition of bacterial repetitive motifs leads to activation and initiation of the immune response. Borrelia lipoproteins signal eukaryotic cells through a heterodimer of TLR1 and TLR2 (Hirschfeld et al., 1999). Studies of TLR2-deficient mice have shown an impaired ability to clear B. burgdorferi. However, loss of the receptor did not alter the inflammation or the arthri-tis severity indicating that the inflammatory response occurred independently of TLR2 (Wooten et al., 2002). To rule out in-volvement of other TLR’s, mice deficient in the key adaptor pro-tein MyD88, which is involved in the signal transduction uti-lized by most of the TLR’s, were experimentally infected. How-ever, this also resulted in the same observation as in TLR2-deficiency, and even higher pathogen load in tissues (Bolz et al.,

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2004; Liu et al., 2004). It became clear that MyD88 signaling is necessary for pathogen clearance, but not for induction of the inflammatory response and disease progression. MyD88 signal-ing has also been demonstrated to be involved in RF resolution, as B. hermsii-infected mice had extremely high spirochetemia but still could respond with IgM production. The antibody re-sponse was, however, delayed and lacked reactivity to one anti-gen, indicating that although the mice could recognize B. herm-sii, they may have a defect in the ability to rapidly respond to new serotypes (Bolz et al., 2006). As the inflammatory response and the adaptive immune response seem to be functional de-spite the loss of TLR signaling pathway, it raises the question if it is the cells of the early innate response that fail to initially control the infection. Bone-marrow derived macrophages from MyD88-deficient mice could kill B. hermsii as well as wild-type macrophages; however, the kinetics was slow. Opsonization of the bacteria improved the kinetics indicating that the deficiency affected the rate of killing (Liu et al., 2004). In addition, degra-dation of the bacteria when they were internalized did not occur (Bolz et al., 2006). In contrast, Shin and co-workers found no defect in the degradation of B. burgdorferi, instead the uptake of the bacteria was impaired (Shin et al., 2008). The discrepan-cy may be an effect of the use of macrophages from different origins, or by the different methodologies. Although it is unclear how a deficiency in TLR signaling affects the ability of mice to limit bacterial growth, phagocytic cells may be involved. As many of their functions are induced by phagocytosis, a slight defect in the capacity to phagocytose may influence down-stream events such as cytokine and chemokine production. Considering the rapid entrance and growth of RF Borrelia, it is crucial to limit them before they reach high numbers, so ineffec-tive phagocytosis during early infection may result in the high

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and persistent bacteremia observed in the absence of TLR sig-naling.

The balance between pro- and anti-inflammatory me-diators

Many cytokines have been demonstrated to be involved in dis-ease resolution and with pathology seen in both LD and RF. The balance between Th1 and Th2 cytokine production has been suggested to be involved in regulating Lyme arthritis severity, where Th2 cytokines are protective (Matyniak & Reiner, 1995). However, mice deficient in IL-4 do not produce a more severe arthritis (Potter et al., 2000), indicating involvement of mul-tiple factors. The balance between pro- and anti-inflammatory mediators has been extensively studied in LD. IL-10 is a major anti-inflammatory cytokine that has a down-regulatory effect on many inflammatory responses such, as IL-12 and TNF-α pro-duction, and macrophage activation (Couper et al., 2008). IL-10 has been demonstrated to be a cytokine induced in both LD and RF infection both in vivo and in vitro (Gelderblom et al., 2007b; Giambartolomei et al., 1998; Lazarus et al., 2008). The observation that different inbred mouse strains display varia-tions in arthritis severity, despite similar bacterial load in the joint tissue, raises the question of whether a genetically deter-mined balance between pro- and anti-inflammatory cytokines can influence the infection outcome. Brown and co-workers demonstrated that macrophages from the resistant mouse strain produced IL-10, whereas the susceptible mouse strain was a poor IL-10 producer. Instead, these mice displayed a higher level of pro-inflammatory mediators. As elimination of IL-10 in the resistant strain also leads to more severe arthritis and a substantial decrease in bacteria in joint tissue, it was clear that the balance between inflammatory cytokines and IL-10 was

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important in the disease progression (Brown et al., 1999). Bet-ter control of pathogen load in the absence of IL-10 has also been demonstrated in other tissues, probably as a result of a lo-wered threshold for innate immune activation, as demonstrated by higher pro-inflammatory cytokine production (Lazarus et al., 2006). Macrophages respond to viable B. burgdorferi with an increase in IL-10 production and a down-regulation of pro-inflammatory mediators (Lazarus et al., 2008). Evidently, en-hanced IL-10 production may be induced by Borrelia as a strat-egy to prevent an inflammatory response. The fact that B. burg-dorferi load is higher in the presence of IL-10 is indisputable, however, the opposite has been demonstrated in RF. High doses of exogeneous IL-10 reduced pathogen levels in the B. turicatae mouse model (Gelderblom et al., 2007b), as well as ameliorated clinical disease. IL-10 deficiency resulted in increased TNF-α level, higher spirochetemia and higher mouse mortality. The in-ability of these mice to control the bacterial load was suggested to be a result of TNF-α-induced apoptosis of immune cells, lead-ing to a decrease in phagocytic cell numbers (Londono et al., 2008). These experiments have been done in antibody-deficient mice that develop very high and sustained spirochetemia. As antibodies are considered to be of the greatest importance for resolution of RF, their removal may unmask other mechanisms that work as additional defenses during exceptionally high pa-thogen load. It is also possible that the balance between the pro- and anti-inflammatory responses acts differentially between RF and LD. In conclusion, variations in the balance between pro- and anti-inflammatory mediators can change the course of the disease depending on the type of Borrelia. The clinical symp-toms may improve, at the expense of efficient bacterial clear-ance, but they may also worsen, if the pro-inflammatory re-sponse is too strong, even though the bacteria are eradicated.

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Borrelia in the brain

Both RF and LD affect the CNS, causing various neurological symptoms, called neuroborreliosis (Cadavid & Barbour, 1998; Pachner et al., 2001). B. burgdorferi was demonstrated to pers-ist in the brain of non-human primates (Pachner et al., 1995a; Pachner et al., 1995b) and Lyme neuroborreliosis (LNB) has thereafter been demonstrated as a persistent infection, with on-ly minor inflammation (Pachner et al., 2001). In RF, residual brain infections occur when Borrelia persists in the brain with-out being detected in the circulation. Bacteria in residual infec-tion are sensitive to sera from the animal from which they were recovered, and they cannot re-enter the blood, pointing towards the brain as a site were circulatory antibodies have no access (Cadavid & Barbour, 1998). The local brain immune response to RF Borrelia is, however, not as well understood. Although it is considered to be an immunoprivileged organ, the brain is still capable of mounting an immune response through resident mi-croglia cells and to a lesser extent by infiltrating immune cells (Kielian et al., 2004; Olson & Miller, 2004). In the B. turicatae mouse model, it was found that there was no difference in the persistence of the spirochetes in the brain between mice defi-cient in TLR2 and WT mice. Furthermore, microglia cells re-sponded to the infection independently of TLR2, indicating that TLR2 deficiency did not increase the susceptibility to persistent brain infection (Cadavid et al., 2006). TLR signaling has, how-ever, been implicated in having a role in the inflammatory re-sponse in LNB. TLR expression is induced in microglial cells and astrocytes upon B. burgdorferi stimulation in vitro, along with production of pro-inflammatory mediators (Bernardino et al., 2008; Rasley et al., 2002).

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Upon microscopic examination of RF-infected brain tissue, the most common finding is an infiltrate of mononuclear cells (Ca-david & Barbour, 1998) spanning from a low grade inflamma-tion (Cadavid et al., 2001; Shamaei-Tousi et al., 1999) to a more substantial infiltration cells of the macrophage/microglia type (Andersson et al., 2007; Gelderblom et al., 2007a; Sethi et al., 2006). Although a substantial macrophage expansion can be demonstrated during acute brain infection with RF Borrelia, it is still clear that bacteria are not eliminated (Andersson et al., 2007), possibly leading to the persistence observed later (Lars-son et al., 2006b). In vitro, microglia can phagocytose B. burg-dorferi without opsonization (Kuhlow et al., 2005), but there is no in vivo evidence that they actually can clear an ongoing brain infection. Since many molecules such as antibodies and antibio-tics have only limited access to the brain, it is tempting to spe-culate that the brain is used by the bacteria to evade elimination in the circulation and prolong their presence in the host. B. dut-tonii has been demonstrated in mouse brain tissue up to nine months post-infection (Larsson et al., 2006b). The bacteria did not enter a dormant state in the brain since they were killed by ceftriaxone (Larsson et al., 2008), an antibiotic that can pene-trate the BBB and only kills actively growing bacteria (Kazragis et al., 1996; Wormser et al., 2000). Surprisingly, no host genes in the tissue were affected, and immune suppression of the host caused the bacteria to re-enter the circulation pointing towards a silent infection that is confined to the brain by the circulatory immune response (Larsson et al., 2006b). However, upregula-tion of genes involved in the immune response have been dem-onstrated in the B. turicatae model (Gelderblom et al., 2007a). Therefore, differences in gene expression may be influenced by different host states and difference in bacterial virulence. Still, Borrelia species tend to cause persistent infection in the brain,

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which may be a process of evading elimination by the immune system. This will prolong their presence in the host and increase the probability of successful transmission to the next host.

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Aims of the thesis 

 

RF Borrelia is a group of invasive pathogens that can cause a persistent infection, indicating that the host immune response fails in its elimination. The immune response can eliminate the infection as well as worsen the disease. The general aims of this thesis have been to investigate host-pathogen interactions dur-ing RF, with emphasis on the host immune response.

The specific aims were to:

• Investigate the early brain immune response to B. crocidurae infection (Paper I)

• Investigate the pathobiology behind persistent B. duttonii infection (Paper II)

• Evaluate pathology involved in B. duttonii-induced pregnancy complications (Paper III)

• Compare immune response between pregnant and non-pregnant mice during RF infection (Paper IV)

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Results and Discussion 

Paper I 

 

In situ brain response in brain and kidney during early relapsing fever borreliosis

Neuroborreliosis is a consequence of Borrelia invading the CNS (Cadavid & Barbour, 1998). Many RF species are highly capable of invading the CNS (Cadavid et al., 1993; Larsson et al., 2006b; Nordstrand et al., 2001; Shamaei-Tousi et al., 1999). The brain is in many respects considered to be an immuno-privileged or-gan due to limited access of antibodies and immune cells re-stricted by the BBB (Hawkins & Davis, 2005). Still, the brain has its own resident immune system composed of microglia cells, and a limited infiltration of circulatory immune cells (Kie-lian, 2004; Olson & Miller, 2004). Borrelia can establish a per-sistent infection in the brain (Cadavid et al., 2006; Larsson et al., 2006b), pointing towards a failure of the brain immune re-sponse to eliminate the bacteria. In paper I, we wanted to study the early in situ immune cell response and subsequent clearance of the bacteria in the brain and compare it with the kidney, an organ that lacks endothelial cell tight junctions and has unli-mited access to blood-borne immune mediators. The spirochet-al load and immune cell expansion and duration were followed over 14 days in both the brain and the kidney of B. crocidurae infected immune-competent mice. We suggest that the brain is dominated by an innate immune response, and that is not suffi-cient enough to eliminate RF Borrelia.

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Spirochetes are not eliminated in the brain despite a robust macrophage and neutrophil infiltration

During the 14 days we followed the spirochete count in the two organs, a main difference observed was the lack of total elimina-tion of the bacteria in the brain tissue, in contrast to complete elimination in the kidney at day 12 post-infection (p.i.). As re-ported previously (Nordstrand et al., 2001), spirochetes could be found in brain parenchyma two days after the inoculation and reached their highest numbers at day 5 p.i., whereafter the numbers slowly decreased. In contrast, the invasive pattern in the kidney mirrored roughly the pattern of spirochetemia, which had an initial high peak at day 5 p.i., and subsequently receded. The persistence of the spirochetes in the brain, but not in the kidney, may reflect a limited access of immune cells to the brain. However, cells of the microglia/macrophage type ex-panded also, reaching their highest numbers at day 12 p.i. the-reby outnumbering the spirochetes by 20 to 1. Macrophages were present in high numbers in the kidney pre-infection, and following infection, their numbers increased and mirrored the spirochete temporal pattern. The second most abundant cell type expanding in the brain tissue was the neutrophil which ex-panded rapidly, roughly mirroring the spirochetal burden in the tissue. As neutrophils were undetectable pre-infection, the brain is highlighted as an organ that is inaccessible to immune cells of the circulation in the absence of brain infection. However, fol-lowing infection, neutrophils could enter the brain, consistent with the function of the BBB, to restrict passage of cells from the circulation, and become “leaky” upon inflammation or injury (Persidsky et al., 2006). Previously, it has been demonstrated in vitro that B. crocidurae promotes neutrophil migration through endothelial cells (Shamaei-Tousi et al., 2000), so it is likely that the spirochetes also can affect neutrophil migration across the

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BBB. It is also noteworthy that at day 14 p.i., there are still sig-nificantly more neutrophils in the brain than at pre-infection, possibly reflecting the persistence of the bacteria. A substantial number of neutrophils were present in the kidney pre-infection, and these cells did not expand significantly following infection. Their abundance in the kidney pre-infection would allow them to kill Borrelia immediately, without further infiltration, appar-ently mounting a more rapid response than in the brain. Neu-trophils are not efficient at phagocytosis of unopsonized Borre-lia, but can kill them extracellularly (Montgomery et al., 2002). So it is possible that the kidney has an advantage over the brain, as neutrophils may contain the early infection until a more po-tent response can occur. On the other hand, it is well known that macrophages and microglia readily kill and phagocytose Borrelia spirochetes in vitro (Kuhlow et al., 2005; Montgomery et al., 2002), so their failure to clear bacteria in the brain may depend on insufficient activation.

Early defense against B. crocidurae infection of the brain does not involve B- or T-cells

Antibodies are important for Borrelia elimination (Alugupalli et al., 2003a), but they cannot penetrate the BBB. Instead, they can be produced intrathecally by B-cells recruited from the pe-riphery (Anthony et al., 2003; Meinl et al., 2006), a feature which has been demonstrated in the LNB mouse model (Li et al., 2006). In our infection model, essentially no B-cells were detected in the brain until day 12 p.i. Compared to the rapid in-crease of B-cells in the kidney peaking at day 5 p.i., it is clear that B-cells have minimal involvement during early infection in the brain, a feature that may allow the bacteria to evade elimi-nation. B-cells are of the greatest importance in resolving RF (Connolly & Benach, 2001), so the deficiency of B-cells in the

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brain during RF infection may be one of the reasons the spiro-chetes are never completely eliminated in this tissue. As T-cells are also not involved in the brain immune response, we draw the conclusion that during B. crocidurae infection, the innate, rather than the adaptive immune response dominates in the brain. The adaptive immune response is usually initiated after more than four days (Janeway, 2005), so involvement of B- and T-cells should have been evident if it had been engaged. The li-mited involvement of adaptive immunity likely contributes to Borrelia survival, and may clear the way for persistent RF Bor-relia infection.

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PAPER  II 

 

Persistent brain infection and disease reactivation in relapsing fever borreliosis

It has been demonstrated that RF-infected brain tissue can be infectious even when no bacteria can be detected in the blood, pointing towards the brain as a site where Borrelia can reside without being eliminated. Persistent infections up to three years have been demonstrated in animals (Cadavid & Barbour, 1998). Experimental animal brains have also in the past been used to maintain RF Borrelia in the laboratory (Felsenfeld, 1971), indi-cating that the brain immune response is not sufficient to elimi-nate the bacteria. In this study, we show that B. duttonii, in con-trast to the other RF species we investigated, could produce a long-term persistent infection in the brain, without provoking any host response. The bacteria could also re-enter the circula-tion after systemic immune suppression, and grow to almost the same density as the initial spirochetemia. We suggest that the brain, as an immunoprivileged tissue, may be a “safe room” for B. duttonii where it can escape the systemic immune response and persist, so that they can re-enter the circulation when the immune pressure has subsided.

B. duttonii is more persistent in blood and brain than other RF species

Immunocompetent mice were infected with B. turicatae, B. hermsii, B. crocidurae and B. duttonii, respectively, and spiro-chetemia was monitored daily. During the acute phase, great variations in the spirochetemia were observed. B. hermsii-, B.

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crocidurae-, and B. duttonii-infected mice all displayed high initial peak spirochetemia, whereas only 50% of the B. turica-tae-infected mice had a mild first peak, suggesting differences in virulence between species. A great variability in the occur-rence of relapses was also observed between the species. The old world species B. duttonii and B. crocidurae caused more re-lapses, and with higher amounts of bacteria in each relapse, compared to new world B. hermsii. This is consistent with the tendency of old world species to cause more severe disease than new world species (Southern & Sanford, 1969). B. duttonii-infected mice presented a far more persistent spirochetemia than the other species, for the most part constantly spiroche-temic for 50 days p.i. However, after 80 days most of the ani-mals were negative for spirochetes in the blood. We investigated the presence of bacteria in the brain and found moderate num-bers of viable B. duttonii in 90% of the animals at day 80 p.i. Even after 270 days, 20% of the mice still had a persistent infec-tion. In contrast, B. crocidurae was found in the brain at day 25 p.i., but not thereafter, and B. hermsii did not produce any brain infection. As the brain is considered to be an immunopri-vileged organ, the possibility of persistent infection was also in-vestigated in kidney and testis, the former being an organ that is easily accessed by blood components and bacteria, and the lat-ter being an organ with immunological properties similar to the brain. With the exception of B. crocidurae, which was found in testis at day 25 p.i., no bacteria could be found in these organs after they had disappeared from the blood. In this study, B. duttonii was the most neurotropic and persistent species of those investigated, indicating that the brain is the main organ in which it resides after elimination from the circulation. In con-trast, B. turicatae did not cause any considerable blood infec-tion, and was therefore excluded from further experiments.

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However, a certain serotype of B. turicatae has been demon-strated to frequently cause residual brain infection in immune deficient mice and to a lesser degree in immune competent mice (Cadavid et al., 2001; Cadavid et al., 2006). We isolated, how-ever, several different serotypes from the brains of mice, indi-cating that B. duttonii does not display one particular serotype which is preferentially selected for in the brain.

Persistent bacteria can be induced to re-enter the circulation with immune suppression

The natural host reservoirs for B. crocidurae and B. hermsii are rodents, whereas B. duttonii, with a few exceptions (McCall et al., 2007), only has been isolated from man and its tick vector, a vector that predominantly feeds on man. The implicated human specificity of B. duttonii made us ask whether the brain could be a reservoir for the bacteria, where it is protected from, but also restrained by, the circulatory immune response. By immuno-suppression of the persistently infected animals with methyl-prednisolone, we could re-initiate the blood infection in some mice almost to the same density as the initial spirochetemic peak. This indicates that the circulatory immune response re-stricts the bacteria to the brain, a site where the bacteria is un-der less immunological pressure. Gene expression in the persis-tently infected brains was analyzed using a cDNA microarray. Surprisingly, no differentially expressed genes were detected when compared to uninfected mouse brains, pointing towards a persistent infection that is unnoticed by the host brain. Howev-er, as a consequence of persistent B. turicatae brain infection in B-cell deficient mice, some genes involved in the immune re-sponse or immune response-related systems were upregulated (Gelderblom et al., 2007a). Therefore, we cannot completely rule out that the brain response in our model was completely

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silenced throughout the infection. The discrepancy herein may be a consequence of different experimental conditions, since our gene expression study was done in immune competent mice, and performed five weeks later p.i., and with a more brain-adapted RF strain. In summary, we propose that B. duttonii, a RF species suggested to be human specific may use the brain as a reservoir. The low immunological pressure in this tissue com-pared to other physiological niches confines the bacteria. Occa-sionally, when the conditions allow it, Borrelia can re-enter the circulation and potentially be transmitted to new hosts.

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Paper III 

 

Pregnancy complications and transplacental transmis-sion of relapsing-fever borreliosis

In RF endemic areas in Sub-Saharan Africa, pregnant women who become infected with B. duttonii are at risk for pregnancy complications, spontaneous abortions, and neonatal death. Some case studies indicate that pregnant women display higher spirochetemia and more severe disease (Barclay, 1990; Bras-seur, 1985; Dupont, 1997; Jongen, 1997; Mayegga et al., 2005), consistent with the general belief that pregnancy leaves an indi-vidual more susceptible to certain infections. We wanted to in-vestigate the impact of B. duttonii infection during pregnancy, and therefore developed a murine model using immune compe-tent C3H/HeN mice. Human and murine gestational states are quite similar as both have a hemochorial placenta, with a simi-lar blood-placenta barrier (BPB). The BPB is a barrier between the maternal and the fetal circulation where exchange between their circulations occurs (Adamson et al., 2002; Georgiades et al., 2002). The human and the murine gestational time-span can seem to be inconsistent with one another (40 weeks and 19-21 days, respectively), but similarly to humans, the murine ges-tational period is divided into developmental periods, similar to trimesters in humans. Previously we infected mice before pla-centation was finalized. This resulted in a low frequency of con-firmed pregnancies. Thus, in this model, pregnant dams were infected at E9, a time-point when the placentation is completed leaving the infection to proceed over nine days of gestation. In this study, we demonstrate that gestational infection with B.

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duttonii commonly causes growth retardation of the fetus, probably as a consequence of impaired oxygen and nutrient transport as the infected placentas often were observed to be damaged. We could also demonstrate that the bacteria can pass the BPB, and infect the fetus. Interestingly, in contrast to re-viewed case studies, we could demonstrate an ameliorated dis-ease in the pregnant dams, as non-pregnant animals displayed higher spirochetemia, more severe disease, and a higher mortal-ity rate. We propose that RF caused by B. duttonii is detrimen-tal for the fetus, but to a certain degree improves maternal dis-ease.

B. duttonii infection causes destruction of the placenta, result-ing in growth retardation of the fetus

Generally, pregnant mice displayed the same symptoms of the disease as reported in human cases, with high grade spiroche-temia, peaking at day 5 p.i. Gross examination of placentas re-vealed hemorrhaging and indications of tissue necrosis in 66% of the infected placentas, whereas merely 10% of the uninfected placentas displayed the same appearance. Microscopic exami-nation showed additional pathological signs of inflammation manifested as fibrin deposits and leukocyte infiltration, predo-minantly by neutrophils. Spirochetes were frequently detected throughout in the labyrinth area of infected placentas at day 9 p.i., although the mice at this time-point did not exhibit any high spirochetemia, indicating that the pathology demonstrated is bacterially induced. All fetuses were also examined and the main finding was a lower weight of fetuses from infected dams, but no evidence of increased fetal death was observed. Notably, fetuses from infected dams were more frequently displaying pa-thological signs such as hemorrhages and signs of impaired blood flow. The growth retardation was also correlated to a

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higher bacterial load in maternal circulation, pointing to a di-rect association of spirochetes with the fetal outcome. We draw the conclusion that the growth retardation of infected fetuses was a consequence of tissue destruction of the placenta, impair-ing its function in providing the fetus with necessary nutrients. Additionally, a lowered blood hemoglobulin (Hb) value in the infected dams worsened the effect, leaving the fetus with insuf-ficient amount of iron and oxygen.

B. duttonii can pass the blood-placenta barrier and cause intra uterine infection of the fetus

Some case studies have implicated that prenatal infections oc-cur, resulting in a newborn with an ongoing RF infection. As Borrelia frequently can penetrate the BBB (Cadavid & Barbour, 1998; Larsson et al., 2006b; Nordstrand et al., 2001) and the blood-testis barrier (Shamaei-Tousi et al., 2001), barriers with functions similar to that of the BPB, we found this plausible as we also observed a great number of spirochetes in the labyrinth area of the placenta. We therefore determined the rate of trans-placental transmission of the spirochete in the fetus from the infected dams. Amazingly, by microscopic examination, we could detect viable spirochetes directly in fetal organ homoge-nates. By inoculating naïve mice with these organ homogenates, we could demonstrate that 73% of all fetuses inspected had a confirmed infection. The confirmed transplacental transmission of the spirochete opens up considerations about the mechanism for passage through the BPB, and its advantage for the bacte-rium. We have previously demonstrated that B. duttonii invades the brain, a tissue with low immunological pressure (Andersson et al., 2007; Larsson et al., 2006b), possibly as a way of pro-longing their presence in the host and increasing the probability of transfer to a new host. It is therefore tempting to speculate

~ 63 ~ 

 

that invasion of fetal tissue is an additional mechanism ex-ploited by the bacteria for improving the transmission frequen-cy.

Pregnancy ameliorates the severity of RF

Based on human case studies, we predicted that the pregnant condition should exacerbate the infection; however, the oppo-site was observed in the mouse model. Non-pregnant mice de-veloped an almost 6-fold higher spirochetemia than their preg-nant counterparts, as well as severe signs of disease, such as ruf-fled fur, conjunctivitis, dehydration and lethargy. We could also demonstrate a lower Hb concentration in the non-pregnant mice than in the pregnant mice, further aggravating the condi-tion. Mice displaying the most severe spirochetemia even died at day 6 p.i. These physiological symptoms were never observed in the pregnant dams, which clearly were in a better state. As pregnant dams could control the bacterial load much more effi-ciently than the non-pregnant mice, we suggest that the immu-nological response during pregnancy is better capable of clear-ing the bacteria, as well as providing protection for the conse-quences of inflammation. Similar alleviation of disease severity has been demonstrated in Lyme arthritis: arthritis improves in pregnant mice, which is suggested to be a consequence of a shifted cytokine response (Moro et al., 2001). Pregnancy induc-es a shift in the immunological configuration, favoring anti-inflammatory cytokine production in order to protect the fetus from the maternal immune response (Druckmann & Druck-mann, 2005). In summary, we suggest that RF during pregnan-cy does not detrimentally affect the pregnant host, as they are better in controlling the bacterial load and also demonstrate less severe disease symptoms. Instead it is the fetus that suffers from severe consequences as a result of bacterial invasion of the

~ 64 ~ 

 

placenta and the fetal tissue. We also propose that a shifted immune response during pregnancy makes the host less sus-ceptible to RF infection.

~ 65 ~ 

 

Paper IV 

 

Enhanced inflammatory response to relapsing fever during pregnancy

The finding that pregnant animals suffered from a milder RF than their non-pregnant counterparts, made us ask whether a differential immune response between the two conditions could be the explanation. As the host immune response is changed to favor innate immunity over cell-mediated immunity for fetal protection during pregnancy (Druckmann & Druckmann, 2005; Sacks et al., 1999), differences can be examined to reveal im-munological mediators that favor a successful resolution of the disease. B. duttonii are blood-borne and they reach their highest density in blood within 4-6 days after inoculation in the mice model (Larsson et al., 2006a). During this time-span, the adap-tive immune response with specific IgG antibodies has not yet evolved, so early control of the bacteria is dependent on the in-nate immune system. The spleen is the lymphoid organ that is specialized in filtering the blood from debris, senescent erythro-cytes, and blood-borne pathogens, and is also involved in im-mune responses to these pathogens (Cesta, 2006; Janeway, 2005). In this paper, unless indicated otherwise, data was col-lected from analysis of spleen tissue. We suggest that pregnant animals are more competent in clearing B. duttonii from the circulation during the first week of infection, as they exhibit an enhanced pro-inflammatory response demonstrated by in-creased expression of pro-inflammatory cytokines, and greater and earlier infiltration of neutrophils. Furthermore, indepen-dent of the conditions, macrophages were the dominant cell

~ 66 ~ 

 

type, whereas lymphocytes were not involved. We speculate that the pregnant host can establish an early and rapid activation of the innate immune response, whereas non-pregnant animals do not, leading to failure of innate immune cells to keep bacterial numbers under control until a specific and directed immunity develops.

B. duttonii infection affects spleen architecture and immune cell distribution

Consistent with our previous study (Larsson et al., 2006a), the non-pregnant animals developed a very high and prolonged peak spirochetemia, and experienced more severe symptoms of disease than their pregnant counterparts. Antibodies of the IgM class have been proven to be important for successful control of the spirochetemia (Alugupalli et al., 2003a). We could demon-strate that the failure of non-pregnant mice to keep the bacterial numbers low was not dependent on an impaired IgM produc-tion, since a robust IgM response was evident in both condi-tions. Spleen tissue sections were analyzed for spirochetes, and immune cells. Spirochetes could be demonstrated in all com-partments of the spleen throughout the infection, independent of the condition, and many of them appeared to be intact, indi-cating that they were not degraded by phagocytic cells.

Morphological and immunohistochemical analysis of immune cell distribution revealed a change in the proportion of red pulp and white pulp as the infection proceeded, leading to a dimi-nished white pulp and an increase in red pulp. Macrophages were the most abundant cell type in the red pulp, consistent with their known function in this compartment for removal of blood-borne debris and pathogens (Cesta, 2006). Quantification showed that these cells increased in both conditions, but to a

~ 67 ~ 

 

significantly higher degree in non-pregnant animals, possibly as a function of the higher bacterial load in the non-pregnant host. Macrophages are potent killers of Borrelia (Montgomery et al., 2002), so the question that arises is why the non-pregnant mice fail to control the bacteremia, despite the substantial macro-phage infiltration, whereas the pregnant mice are successful in the resolution.

Neutrophils were the second most abundant cell responding to the infection, appearing earlier and with significantly higher numbers in pregnant mice at day 4 p.i., pointing towards a de-layed attraction of neutrophils in non-pregnant mice. As neu-trophils can kill Borrelia extracellularly (Lusitani et al., 2002), an early appearance may give the pregnant host extra control over pathogen load, keeping the early bacterial growth within a certain limit.

As the red pulp compartments seemed to expand at the expense of the white pulp that diminished following infection, we ana-lyzed lymphocyte populations by FACS analysis. Interestingly, there was no evident expansion of B- and T-cells in either of the conditions. Non-pregnant mice even displayed a substantial de-cline in B- and T-cell frequencies that continued to decrease throughout the days investigated, whereas during pregnancy splenic B- and T-cells were unchanged. In conclusion, as lym-phocytes did not respond during the time-span investigated, we suggest that the adaptive immune response is not involved in controlling the B. duttonii infection in the spleen during the first eight days of infection.

~ 68 ~ 

 

The balance between pro- and anti-inflammatory mediators

Non-pregnant mice suffered from high spirochetemia despite great expansion of macrophages in the spleen. Furthermore, a delay in neutrophil appearance compared to their pregnant counterparts, may indicate lower attraction and activation of these cells in non-pregnant mice. A cytokine array analyzing 21 different murine chemokines and cytokines exposed a few in-flammatory mediators in the serum that were differentially ex-pressed between the conditions. Their expression in the spleen was further investigated by real-time RT-PCR. Firstly, CXCL1 (KC), a potent neutrophil chemokine was induced almost 10-fold following infection in the pregnant mouse spleen, and re-mained up-regulated throughout the infection, whereas it re-mained unchanged in non-pregnant animals. The importance of CXCL1 in attracting neutrophils has been demonstrated in LD infection models, as production of CXCL1 correlates with great-er control of bacterial numbers by neutrophils (Xu et al., 2007). Thus, higher expression of this cytokine during pregnancy may explain the higher infiltration of neutrophils, and subsequently better control of bacterial growth. Secondly, CCL5 (RANTES), a T-cell chemokine, was highly expressed in the pregnant mice at day 2 p.i. possibly explaining the observation that T-cell num-bers remained at pre-infection level in the pregnant spleen, whereas they decreased in the non-pregnant spleen. Thirdly, monocyte chemotactic protein 1 (MCP-1), was the most highly expressed chemokine independent of conditions, consistent with the dominance of macrophages following infection. How-ever, non-pregnant mice had significantly higher MCP-1 expres-sion at day 2 p.i., which may explain the greater expansion of macrophages in non-pregnant mice.

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As cytokines can enhance or reduce the function of immune cells, we examined the local expression of some pro- and anti-inflammatory cytokines. Pregnant animals induced a signifi-cantly higher expression of IL-6, IL-12, and IFN-γ at day 2 p.i., in contrast to the non-pregnant animals, who displayed only a weak expression of these cytokines at the same time-point. IL-6 stayed highly expressed in the pregnant animals throughout the infection. On the other hand, TNF-α, which is a potent pro-inflammatory cytokine was not expressed at the same high le-vels as the former cytokines, but reached significantly higher expression in the pregnant mice at day 6 p.i. Surprisingly, non-pregnant animals did not up-regulate this cytokine at all, which could be expected considering the high numbers of macrophag-es that were present in their spleens. TNF-α is usually expressed at high levels by activated macrophages (Janeway, 2005), so the lack of expression in non-pregnant animals may be due to im-perfect activation of these macrophages, despite the high num-bers of bacteria present in the spleen. A pro-inflammatory re-sponse must be balanced with an appropriate anti-inflammatory response, which also could be demonstrated in all animals. Pregnant animals had a strong induction of IL-10 ex-pression, peaking at day 6 p.i., simultaneous with the expres-sion of pro-inflammatory mediators decreasing. However, the non-pregnant animals also had robust up-regulation of IL-10, despite the lack of expression of inflammatory mediators. IL-10 is a multifunctional cytokine that can inhibit macrophage func-tions, and also limit and terminate inflammatory responses, playing an important role in the balance between induction and down-regulation of inflammatory responses (Couper et al., 2008). Furthermore, IL-10 has been implicated in reducing a detrimental inflammatory response to Borrelia infection at the expense of efficient bacterial control. (Brown et al., 1999; Gel-

~ 70 ~ 

 

derblom et al., 2007b; Lazarus et al., 2008). So it is possible that by inducing IL-10 production, Borrelia will have a powerful mechanism to force the host to dampen the inflammatory re-sponse.

sTNF-α RI is the receptor for TNF-α, but in normal immune surveillance, it can be shed from cell surfaces and thereby inhi-bit the bioactivity of TNF-α (Björnberg & Lantz, 1998). Accord-ing to the cytokine array, sTNF-α RI was the most highly ex-pressed serum protein following infection in both conditions, and an ELISA confirmed that its release was induced, roughly mirroring spirochetemia. In the pregnant mice, this makes sense as they also exhibited an increase in TNF-α, but in the non-pregnant mice this seems to be unnecessary, unless it is in-duced by the bacterium itself to further dampen the inflamma-tory response.

Generally, pregnancy leads to a shift in the cytokine response (favoring humoral immunity at the expense of cell-mediated immunity), but also an augmentation of the innate immune re-sponse with increased levels of acute phase proteins (Sacks et al., 1999). In our model, early attraction of macrophages and neutrophils, and robust and continuous expression of IL-6, IL-12, and IFN- γ may increase the activation of macrophages, as well as keep the threshold of innate immune response activation low, leading to successful control of bacterial growth. The ex-pression of IL-10 and sTNF-α RI will balance the response to prevent unnecessary damage. This was demonstrated in the pregnant mice, whereas non-pregnant mice had a delayed neu-trophil infiltration, and a cytokine pattern that had a tendency to be only anti-inflammatory. Consequently, non-pregnant an-imals failed to control bacterial numbers even though they at-tracted macrophages to the tissue. We therefore suggest that an

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early pro-inflammatory innate immune response, during the first days of infection, gives the pregnant mice an advantage as they can keep the bacterial numbers within a certain limit until a more specific defense can occur.

~ 72 ~ 

 

Concluding remarks 

The main conclusions presented in this thesis are that RF Bor-relia has have strategies to evade elimination. They can pene-trate and reside in tissues with a weak immune response, al-lowing them to prolong their presence in the host. Outside these protective niches, during the initial infection, they pro-voke an immune response that is not sufficient enough to elim-inate them. The main findings are as follows:

The early brain immune response during RF borreliosis is dominated by innate immune cells. Interestingly, the RF Bor-relia is not eliminated, indicating that the response is insuffi-cient. The failure of elimination is advantageous for the bacteria as it prolongs their presence and may lead to persistent infec-tion.

Persistent infection of brain occurs with low levels of bac-teria. The numbers of bacteria may be too low to provoke an immune response. As immune suppression allowed the bacteria re-enter the circulation, it indicates that the bacteria are con-fined to the brain by the circulatory immune response. We pro-pose that the brain may function as a reservoir for B. duttonii, a bacterium that rarely has been detected in any host other than man.

B. duttonii infection causes serious pregnancy complica-

tions, mainly for the fetus. The main finding was growth retar-dation of the fetus. The bacteria penetrated the blood-placenta barrier frequently, and caused peri-natal infection. Pregnancy ameliorated the severity of the disease and lowered the bacterial

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~ 73 ~ 

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~ 74 ~ 

 

Sammanfattning på svenska

Återfallsfeber är en bakteriell infektionssjukdom som or-sakas av Borrelia spiroketer. Bakterien sprids oftast av Or-nithodoros fästingar, och den kan bara existera i en fästing eller i ett värddjur. Infektionen karakteriseras av återkommande pe-rioder med hög feber, och varierande influensaliknande symp-tom. Dessa orsakas av att bakterien växer i blodet, så kallad spi-roketemi. Bakterien är mycket bra på att penetrera olika vävna-der och organ, inklusive det centrala nervsystemet (CNS). Vi använder olika musmodeller för att studera bakterie-värddjur interaktioner, och då med fokus på patologi och immunologi.

Hjärnan har en blod-hjärn barriär som gör att många komponenter i blodet, till exempel immunceller, har begränsad åtkomlighet. I manuskript I, så jämförde vi bakteriens invade-rings förmåga, med immuncellsexpansionen i hjärnan och nju-re. Njuren har ett vanligt endotellager, så immuncellerna har obegränsad tillgång till detta organ. Vi kunde i detta arbete visa att hjärnans immunsvar dominerades i första hand av mikrog-lia/makrofag celler, och i andra hand av neutrofiler. Trots dessa potenta immuncellers närvaro så blev aldrig bakterierna totalt eliminerade från hjärnan. I njuren kunde ett liknande mönster för immuncellerna påvisas, men till skillnad från hjärnan, så försvann Borrelia totalt från detta organ. Vår hypotes är att Borrelia skyddas från det systemiska immunsvaret i hjärnan, och att detta kan leda till långvarig infektion.

I manuskript II så visar vi att återfallsfeber Borrelia or-sakar långtidsinfektion i hjärnan. Infektionen provocerade inte något immunsvar i hjärnan, eftersom inga skillnader i genut-tryck mellan de icke infekterade och de långtidsinfekterade

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hjärnorna kunde påvisas. Vi visade också att blodinfektionen kunde återaktiveras genom suppression av immunsystemet. Detta tyder på att bakterien är innesluten i hjärnan, som en di-rekt påverkan från immunsvaret i cirkulationssystemet i övriga delar av kroppen. Detta kan anses vara en bra bakteriell strate-gi, eftersom det förlänger den tid bakterien kan stanna i värd-djuret och som därmed ökar chansen för överföring.

Återfallsfeber är en vanlig orsak till komplikationer vid graviditet i Afrika. Med tanke på bakteriens invaderande förmå-ga så studerade vi infektionens påverkan på dräktiga möss. I manuskript III så visar vi att återfallsfeber Borrelia infektionen i huvudsak drabbade fostren. Det vanligaste fenomenet var låg fostervikt. Eftersom många placentor uppvisade patologiska tecken, så tror vi att detta orsakar otillräckligt syre- och närings-transport till fostren. Vi kunde också påvisa att bakterien kunde tränga igenom blod-placenta barriären och infektera fostret re-dan innan födseln. De dräktiga mössen hade överraskande nog lägre spiroketemi, och uppvisade mildare sjukdomssymptom. Vi tror därför att immunsvaret under dräktigheten är bättre läm-pat att kontrollera spiroketernas tillväxt.

Den påvisade skillnaden i spiroketemi och sjukdoms-symptom i manuskript III fick oss att undersöka immunologis-ka skillnader mellan de två olika tillstånden under infektionen. I manuskript IV så fokuserar vi på mjälten, som är ett immunolo-giskt organ inbegripet i att bekämpa blodinfektioner. Vi kunde i detta arbete visa att generellt, under båda tillstånden, så var makrofager de vanligaste cellerna som svarade på infektionen. Dräktiga djur hade förutom makrofager, dessutom en tidig neu-trofil expansion, vilket inte kunde påvisas i de icke dräktiga mössen. Vi undersökte kemokin och cytokin uttrycket i dessa djur. Dräktiga djur hade högre uttryck av pro-inflammatoriska

ctaVtkdmkadl

ShuincDslö

cytokiner, octraherar neutanti-inflammVår slutsats tröskel för dekontroll av dessutom anmössen lederkontroll av danti-inflammdämpar ytterleda till ökad

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matoriska proär att dräkt

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matoriska prorligare den in överlevnad a

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munsvaret, gesom hjärna eer den initialaäckligt starktda till stark r utvecklas. I

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~ 76 ~ 

högre uttryckrraskande noteiner lika m

tigheten verkoriska responn. Återfallsfetoriska medide kontroll av

matoriska resoteiner i det nflammatorisav bakterien.

resultat somer Borrelia uenom att peneller ett fostera infektionent immunsvar tillväxt innaI slutändan sddjuret, vilke

k av ett kemog så uttryck

mycket i bådakar ge en lägnsen vilket ledfeber Borreliiatorer. Hos v bakteriens sponsen. Pr icke dräktigska response

m presenterastvecklat strat

netrera vävnar. Utanför den, så verkar dr för att bli an ett mera så handlar deet ökar sanno

mokin som at-ktes dock tvåa tillstånden.gre aktivitetsder till bättreia inducerar de dräktigatillväxt, samtroduktion avga tillståndeten, vilket kan

s i denna av-tegier för att

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-å . s e r a t v t n

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h -r

 

~ 77 ~ 

 

Acknowledgements  Sven Bergström, my supervisor. It has been a pleasure to be intro-duced to the field of Borrelia. I will always be a “spirochetologist” in my heart. THANK YOU! Betty Guo, my co-supervisor: you have been a great support and are a great friend. Additionally, it is a plea-sure to go out for drinks with you! The Borrelia Group, present and former members, for maintenance of the great spirit of the “Borrelia team”: Christer “Korv” Larsson: We have brought many “daughters to the slaughter” together, however “med ett schysst hjärnrör kan man förändra världen”. Good luck in Baltimore, I will miss you. Ingela Nilsson, thank you for support, your help with my projects, and your friendship. Pär C, it has been a pleasure to get to know you. I really miss you, but NOT your music taste. Thanks to Lelle B, you are such a nice guy, Ignas,”no problem” Bunikis for positivity, Elin Nilsson för “schlagerfrossa and stick handling”, Marie Bonde for finsk sisu, Patrik ”Pats” Engström for being the one to blame on, now when Lelle has left the building”, Lisette Marjavaara, who challenged me with “Vårruset”, Sabrina, Åsa Gylfe for medical care. Special thanks go to Annika Nordstrand. All at the department that have contri-buted to a good spirit, and for making my work easier: “Professor” Stenman: thank you for the appearances of “surprises” in my freezer, Monika W for EXCELLENT music taste and for maintaining the “hästtjej” spirit in me, Jenny L, for being a kind and friendly person that brings “fika” when you least expect it, Kristina Ruth, for teach-ing me how to become a good friend with the FACS-machine. The administrative heroes: Marita, Ethel and Berit for sorting out many problems, Agneta and the rest of the personnel at Lab-Service, per-sonnel at the GDL for coagulation plug inspections, Mariella for teaching me to be a better person, Marek for fixing my old carcass to computer several times, Sonja S for being Sonja, Anna F for immu-nological “pep-talks”, Jenny S and Sara K for being great “compa-

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dres” in immunology course teaching, Micke S for excellent expertise in the stick business, Anna Å: thanks for beer corner maintenance and for support in less important businesses. All people that contributes to a great scientific and social spirit at the department: Maria N, Lisan-dro, Reine, Kerstin M, Stefan N, Annika S, Stina, Barbro, Connie, Monica, Linda, all in “Näs-gruppen”, Edmund, Jonas, Theresa, Barbara, Sofia, Jeanette, Pelle, Christoffer, Martin G, Micke W, Ulrich, Jörgen, Sun, Bernt-Eric, Maria F, and many more…… Tack till vänner utanför institutionen som förgyllt livet: Anna och Sussi, mina bästa vänner sedan länge. Lill-Ellen: alla behöver inte bli hästtjejer, Hasse, du är faktiskt en riktigt schysst kille, Mona, Jona-than, Arne och Ingmari, för Västerås och Rugö, och för att få vara ”del av er familj” också, Filippa och Jerker, Hans-Ola S för drama-tik, Janne T för vänskap och filmtips, Britt, snart är det din tur! An-ders Å för Fredmans Apostlar, Micke N för music quiz, Maud W med familj, Vännäs människor: Erik Å, Flisa, sist men inte minst: Micke Nilsson: Jag ser fram emot dina frågor. Tack till den underbara “familjen” Tannemyr: Maria och Janne: för att ni ”tar hand” om mej i Stockholm, Peppe & Eva, Micke & Helen: Mille gracie, för Celleno och för Sten & Flisa, och för allt annat. Julia och Evelina: håll ut, snart är ni vuxna. Ni kommer att fixa det! Anne-li: Du är bäst! Min familj: De sista åren har varit ganska jobbiga; så mycket har änd-rats. Men vi har hållit ihop, och alltid kunnat se det positiva, och dess-utom det roliga i dessa situationer. Utan er, och ert stöd, hade denna avhandling inte varit möjlig: Monica, Annika, Ille med ”jeans byx-en”, Randie, Johnny, Johan, Malin, Lukas, Simon, Punk-Jocke, Helen, Vidar, Ylva. TACK! Slutligen: Christer: ♥♥♥♥♥♥♥♥♥♥♥♥♥♥♥♥♥♥♥♥♥♥♥♥

   

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