April 2005

Consortium leader: Professor SVEN BERGSTR�M,
Department of Molecular Biology, Ume� University, SE-901 87 Ume�, Sweden, Tel.: +46 (0)90 7856726, [email protected]
Other project leader of the consortium:
Professor Matti Viljanen,
Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland, Tel.: +358-2-3337330, email: [email protected] , homepage: http://www.utu.fi/research/tic/viljanen/
Key words: Borreliosis, immune defense, immune evasion, and reactivation

Abstract
The aim of this project is to gain knowledge of the interactions between Borrelia spirochetes and the host during infection. We are using both the Lyme borreliosis and the relapsing fever borreliosis spirochetes as model organisms. We are investigating how the Borrelia spirochetes can circumvent the immunological defence, how they spread form the infectious focus, reach various sites in the mammalian body, and how the spirochetes can live in these tissues at a dormant state, reactivate, return to the circulatory system and cause acute disease again. We also aim to characterize and define the components involved in the interactions between Borrelia and human cells, including the cells of the innate and adaptive immunity.

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The current status of the project is presented below according to the questions and aims of the original research program

Adhesion of Borrelia to endothelium is mediated by specific integrin binding.

This project will further extended to identify outer membrane proteins, i.e. P66, P13, OspA-C etc in pore formation, adhesion to host cells, and tissue tropism. Structural characteristics of some of these proteins are known and will be used to design inhibitory compounds for the binding and interaction process. Additionally, the Borrelia species that causes relapsing fever binds and aggregates erythrocytes as a possible additional mechanism for evasion of the immune system. A potential adhesin involved in this aggregation has been identified. Pore forming assays and aggregation assays will be used to measure the inhibitory effects of test compounds chemical inhibitors that block the function of Borrelia species. Thus, we have demonstrated in earlier studies that some species of relapsing fever Borrelia adhere to erythrocytes, causing the formation of erythrocyte rosettes. This aggregate of Borrelia and red blood cells may protect the spirochetes and contribute to the delayed immune response seen in rosette-forming strains compared non-rosette-forming strains. Mice infected with a rosette-forming strain exhibited more severe pathology and reduced blood flow compared to mice infected with a non-rosette-forming strain. Therefore, adhesins and receptors involved in this interaction would lead to possible therapeutic tools against relapsing fever. We have now identified a 27 kDa Borrelia protein that binds to a component of human erythrocyte membranes. We are in the process of purifying this protein in order to capture the erythrocyte receptor by affinity chromatography and subsequently identify it by mass spectrometry. A Borrelia library will be constructed and screened using the purified receptor.

Borrelia use host proteases to spread from the infection focus to blood, and to invade distant organs.

The initial studies concerning this part were published in 2001(Nordstrand A., et al. "Delayed kidney and brain invasion by Borrelia crocidurae in plasminogen knock-out mice". Infect Immun 2001, 69: 5832-5839). Further protease evaluation is described below, where B. crocidurae, B. hermsii and B. duttoni are investigated in relation to invasion capability and characteristics. A murine model has been established to test Borrelia-mediated activation of host proteases and modulation of the immune responses in the induction of abortion associated with relapsing fever borrelisosis. Immune cell subsets and the role of proteases are being investigated (Andersson M, Larsson C, Bergstr�m S and Nordstrand A. Invasion, inflammation and host proteases in a murine model of Borrelia-induced complications during pregnancy. 2005 Manuscript in preparation). So far matrix metalloproteases and plasmin have been tested. However, no upregulation have been documented so far, although other proteases will be tested.

Borrelia can evade the first line defense and direct the later specific immune response by interfering with the function of neutrophils and dendritic cells.

We have previously shown that contact with B. burgdorferi induces maturation and IL-8 production of immature dendritic cells (DCs) in a similar manner as contact with LPS, a known maturation inducer. These observations suggest that the interplay between borreliae and DCs is similar to the interplay of DCs with other microbes. However, gene expression studies are essential to investigate in more detail the possibility that borreliae are somehow manipulate DCs to their benefit. We have now completed the microarray experiments where we determined the gene expression profiles of borrelia-stimulated and -unstimulated DCs, and compared the borrelia-induced changes in DC gene expression to the effects of LPS. Changes in gene expression were analysed in four time points, and each time point was done in triplicate resulting in 36 microarray hybridizations with two technical repeats. A computer algorithm has been set up to finalise the array results. As a result, we have identified the differential expression of up to several hundred genes, many of which are important regulators of immune response. Confirmatory experiments with quantitative PCR and protein arrays are underway.

We have also studied the role of borrelial outer surface proteins (Osps) in the phagocytosis of borrelia by neutrophils. In theses studies, we have used B. burgdorferi strains B31 and B313, which is a mutant of B31 lacking OspA and OspB surface proteins, among others. Flow cytometry and Baclight bacterial viability assays have been used to assess the amount and viability of bacteria ingested by neutrophils. We have found that B31 is phagocytized more efficiently than B313. Thus, OspA and/or OspB seem to play a role in the phagocytosis of Borrelia by neutrophils, but this has to be confirmed by using an OspAB complemented B313 strain, which we have just cloned. In addition, we have expanded our experiments concerning Borrelia-neutrophil interaction into a new direction by setting up assays where neutrophil phagocytosis of Borrelia is manipulated with antibodies and other reagents interfering with neutrophil receptors and signal transduction.

Toll-like receptor expression is modulated in immune cells as an effect of spirochete invasion. The Borrelia-TLR interaction is central for clinical outcome.

Organ tissues available have been evaluated by immunohistochemistry (included in PhD project, M Andersson). B. crocidurae, B. hermsii and B. duttoni are evaluated regarding invasion characteristics. Preliminary results indicate differential ability of the species to invade as well as by the invaded spirochetes to trigger an inflammatory response in situ. This will be further investigated by Real-Time PCR methodology.

Severe symptoms are associated with a certain type of T-helper response. Manipulation of the Th1/Th2 ratio and attenuation of inflammatory responses may be used to prevent severe manifestations of borreliosis.

We are currently preparing a manuscript on one part of this project, where we describe the immune response in brain during the early process leading to neuroborreliosis. We report herein on a macrophage-dominated response, with IL-10, IL-15 and IFNg as the most important cytokines. Interestingly following IL-10 increase, the inflammation subsides to a low level, but does not result in eradication of the bacterium from the tissue. These results indicate that during infection IL-10 down regulates the intense macrophage dominated response, but this may occur at the expense of complete eradication of the bacteria (Nordstrand A, Anderssojn M, Shamaei-Tousi A, Jansson A and Bergstr�m S. In situ immune response in brain and other organs at the early stage of murine neuroborreliosis. 2005 Manuscript in preparation).

Whether this mechanism reflects the initial explanation to bacterial persistence or not remains to be addressed, as does the role of IL-15, a cytokine that so far has not been investigated during borreliosis. This will be further investigated by Real-Time PCR methodology.

As a new approach, we have investigated the effects of treatment with anti-TNF-a antibody and/or antibiotics on the development and persistence of borrelia arthritis in mice. The results suggest that the administration of anti- TNF-a with or without antibiotics does not lead to amelioration of arthritis. However, during these experiments we have observed that anti- TNF-a given after ceftriaxone treatment leads to activation of a latent form of borrelial infection. Characterization of the cellular and molecular biology of this latent form of borrelial infection is currently underway.

We have also analysed the effect of immunomodulator Linomide on borrelia arthritis in mice. Linomide caused a mild reduction in joint swelling of borrelia infected mice, but there was no decrease in lymphocyte infiltration in Linomide-treated animals.

Borrelia may, by crossing very tight barriers, such as the blood-testis and blood-brain barriers, invade organs and use these as reservoirs for an extensive length of time. Reactivation of infection can occur from these sites.

Results indicate that B. duttoni are able to reside in the immune privileged organ brain an extensive time after their disappearance from blood, and can be reactivated to appear in blood by immunosuppresing conditions. This appears to be a unique feature of this species. The result from this work is expected to result in submission of a manuscript during 2005. Interestingly, we have found that the RF Borrelia species tested in this project has a great ability to pass the blood-placenta barrier. As many as 70% of the foetus are infection positive d18 after plug formation if infected at day 9 indicating a effective mechanism to also pass this important physical barrier to an immune privileged site.

Publications