Consortium leader: Docent WILLIAM AGACE,
Immunology Unit, Department of Cell and Molecular Biology, Lund University, Sweden, +46-(0)46-2220416, William.Agace@immuno.lu.se
Project partners of the consortium:
Professor Mikael Skurnik, The Haartman Institute, University of Helsinki, +358 (0)9 191 25290, mikael.skurnik@helsinki.fi
Professor Sirpa Jalkanen, Department of Medical Microbiology, University of Turku, Finland, sirjal@utu.fi
Professor Catharina Svanborg, Division of Microbiology, Immunology and Glycobiology, Lund University, Sweden, +46-(0)46 173972, catharina.svanborg@mig.lu.se
Professor Mary Jo Wick, Dept. Medical Microbiology and Immunology, Göteborg University, Guldhedsgatan 10 143 46 Göteborg, tel. 031 342 4602, mary-jo.wick@immuno.gu.se
Key words: Mucosa, epithelium, homing, chemokines
RESULTS
William Agace's group
Participating persons in Agace's group :
Hanna Stenstad, Lund University, Hanna. Stenstad@immuno.lu.se
Nengt Johansson Ph.D., Knut Kotarsky Ph.D.
Key Words:Epithelium, intestine, lymphocyte, bacterial microflora, Laser capture microscopy, Microarray
Results
(i) CCR9 independent effector CD4+ T cell recruitment to the small intestinal lamina propria.Blood (on line-2006) (Hanna Stenstad) We have demonstrated that CD4+ T cells activated in gut associated lymphoid tissue are induced to express a specific range of chemokine receptors and that these cells enter the intestinal mucosa in a CCR9 dependent and CCR9 independent fashion.
(ii)Regulation of CCL25 in small intestinal epithelial cells; Cdx proteins enhance the activity of the CCL25 promotor. 2006. J. Immunol. 176 4642-3651 (Ericsson, Kotarsky). We have previously demonstrated a critical role for CCL25 in the recruitment of T cells to the intestinal mucosa. In this study we examined possible mechanisms regulating the expression of CCL25 in the small intestinal epithelium. Here we demonstrate that CCL25 is not regulated as other inflammatory or homeostatic chemokines, and that the gut flora is not important for driving constitutive expression of CCL25.
(iii) Selective generation of gut-tropic T cells in gut associated lymphoid tissue (GALT); requirement for GALT dendritic cells and adjuvant. J. Exp. Med. 2003. 198; 963-969.
Functional specialization of gut CD103+ dendritic cells in the regulation of tissue selective T cell homing. 2005. J. Exp. Med. 202 1063-1073. (Bengt Johansson-Lindbom). In this line of studies we demonstrate a critical role of intestinal derived dendritic cells in the generation of gut tropic T cells, in particular a subset of CD103+ DCs which we believe come from the intestinal lamina propria. Additional preliminary data using intestinal derived DCs from germ free mice (from the Karolinska institute) suggest that the intestinal flora plays no role in imprinting these DCs with the ability to generate gut tropic T cells.
(iv) Bacterial:Epithelial cross-talk and the establishment of the mucosal immune system. (Knut Kotarsky, Hanna Stenstad). We have isolated small intestinal and colonic epithelial cells (excluding lymphocytes) from the germfree and conventional murine intestine using Laser Capture Microscopy (LCM). RNA was obtained and amplified by repetitive in vitro transcription, and microarray analysis has been performed using affymatrix chips covering the entire mouse genome. Further analysis confirmed that microarrays did not contain contaminating lymphocyte gene products. We have identified a chemokine that is highly expressed in the colon, less so in the small intestine and are currently assessing the role of this chemokine in generation of the colonic immune cell compartment.
(v) CCR9 independent T cell migration to the intestinal mucosa (Hanna Stenstad). Hanna Stenstad has developed a model to generate gut homing T cells in vitro and by transferring these cells into recipient mice is currently addressing the mechanism of effector T cell entry to the colon and CCR9 independent T cell migration to the small intestine
Selected publications:
Johansson-Lindbom, B., Svensson, M., Pabst, O., Palmqvist, C., Marquez, G., Forster, R., Agace, W. Functional specialization of gut CD103+ dendritic cells in the regulation of tissue selective T cell homing. 2005. J. Exp. Med. 202 1063-1073
Ericsson, A., Kotarsky, K, Sigvardsson, M., and Agace, W. Regulation of CCL25 in small intestinal epithelial cells; Cdx proteins enhance the activity of the CCL25 promotor. 2006. J. Immunol. 176 4642-3651
Stenstad, H., Ericsson, A., Svensson, M., Picarello, D., Briskin, M., Marquez, G, and Agace, W. CCR9 independent effector CD4+ T cell recruitment to the small intestinal lamina propria. 2006. Blood. Jan 3 (Epub ahead of print)
'Serum resistance determinants of Yersinia enterocolitica serotype O:3'
Subproject leader: Prof. Mikael Skurnik, University of Helsinki, Haartman Institute, Department of Bacteriology and Immunology, P.O.Box 21, 00014 University of Helsinki, Finland
Tel: +358-(0)9-19126464, Mobile: +358-(0)50-3360981, Fax: +358-(0)9-19126382
E-mail: mikael.skurnik@helsinki.fi
Researchers: Marta Biedzka-Sarek, PhD student
Key words: Yersinia enterocolitica, YadA, Ail, lipopolysaccharide, complement system, factor H
Project Summary:
One task in the studies on the bacterial:host interactions is characterization of the bacterium interactions with serum complement system. Yersinia enterocolitica serotype O:3 (Ye O:3) resists efficiently complement-mediated bactericidal activity of human serum. This serum resistance is multifactorial from the bacterial side including several factors. Two of those factors are outer membrane proteins. YadA, an adhesin, mediates bacterial adherence to extracellular matrix proteins, while Ail is needed for the cellular invasion. Another factors possibly involved in serum resistance are lipopolysaccharide (LPS) O-antigen (O-ag) and the hexasaccharide branch, known as outer core.
(i) Contribution of YadA, Ail and lipopolysaccharide into serum resistance of Y. enterocolitica serotype O:3
The role of YadA, Ail, O- for the survival of Ye O:3 in nonimmune human serum was studied by constructing 23 mutant strains of Ye O:3 expressing different combinations of YadA, Ail, LPS O-antigen and outer core. The ability of the strains to resist complement action was tested in normal serum (with functional classical and alternative complement activation pathways) and in EGTA-Mg treated serum (only alternative pathway functional). Our results showed that the Ye O:3 serum resistance is a multifactorial process. YadA was found to be the most potent single serum resistance factor, however, Ail also appeared to be indispensable for the bacterial survival and its role was related to delaying the killing of bacteria. The LPS O-antigen and outer core appeared to have a minor and often negative effect on serum resistance when in combination with YadA and/or Ail. We also noticed that O-ag as a single factor can efficiently prevent C3 deposition on bacterial surface. No direct correlation between the C3 deposition pattern and bacterial resistance was observed. This part of the project is published in (1).
The efficacy of the complement system depends on its tight regulation. Some complement components function as regulators able to control complement activation. One of these regulatory proteins is factor H. It inhibits the alternative pathway activation thereby protecting the host from the damaging effect of the complement action. Many pathogens, however, have evolved to bind this regulatory protein and avoid complement attack.
(ii) Identification of factor H ligand on Y. enterocolitica serotype O:3
Preliminary experiments indicated that factor H binding by Ye O:3 is YadA-dependent. In order to verify YadA region interacting with factor H, twelve mutants expressing truncated YadA have been constructed. Their resistance to normal and EGTA-Mg nonimmune human serum is currently under investigation.
Collaboration with Jalkanen group
The ability of Vap1 knockout mice to manage enteropathogen infection was tested using Yersinia enterocolitica as a model organism. The results are published in reference (2).
Scientific publications:
- Biedzka-Sarek M., Venho R. and Skurnik M. 2005. Role of YadA, Ail and lipopolysaccharide in serum resistance of Yersinia enterocolitica serotype O:3. Infection and Immunity. In press.
- Stolen, C. M., F. Marttila-Ichihara, K. Koskinen, G. G. Yegutkin, R. Turja, P. Bono, M. Skurnik, A. Hanninen, S. Jalkanen, and M. Salmi.2005. Absence of the Endothelial Oxidase AOC3 Leads to Abnormal Leukocyte Traffic In Vivo. Immunity 22:105-15.
SUBPROJECT: Sirpa Jalkanen's Group
Sub-project: Bacterial: Epithelial cross-talk and the establishment of the mucosal immune system
Project leader: ProfessorSirpa Jalkanen, Department of Medical Microbiology, University of Turku, Finland, sirpa.jalkanen@utu.fi
Researchers: Marko Salmi (National Public Health Institute), Arno Hänninen (University of Turku), Fumiko Ichihara- Marttila (university of Turku), Kaisa Koskinen (University of Turku), Suvi Nevalainen (University of Tuirku)
Results
Leukocyte traffic between the blood and tissues is needed for normal immune homeostasis as well as for mounting adequate inflammatory responses. The spectrum of inflammatory diseases for which the inadvertent migration of leukocytes is proving to be critical is increasing all the time. Therefore, understanding of the mechanisms of how leukocytes leave the blood and enter tissues is critical for the understanding the pathogenesis of inflammatory reaction and for the development of novel anti-inflammatory strategies.
We have previously provided in vitro evidence that antibodies against AOC3 (also known as EC1.4.3.6, placental amine oxidase or vascular adhesion protein-1 (VAP-1)) partially block leukocyte-endothelial interactions suggesting that AOC3 may be involved in leukocyte binding to vessels. This enzyme belongs to semicarbazide-sensitive amine oxidases (SSAO), that catalyze oxidative deamination of primary amines in a reaction producing bioactive aldehydes, hydrogen peroxide, and ammonium as end-products. The enzymatic properties of SSAO have been known for decades, but their physiological functions have remained elusive.
The project has been aiming at resolving the role of vascular adhesion protein-1 (VAP-1)/AOC3 in mucosal immunity and host defence against microbes. VAP-1/AOC3 has been earlier shown to mediate leukocyte trafficking to sites of inflammation for example in arthritis. During the course of the project we were able to create VAP-1/AOC3 knockout mice in pure background and test their mucosal defence system. First of all lymphocyte trafficking to Peyer's patches when measured using intravital microscopy was diminished. Moreover, these mice showed a slight but statistically significant reduction in lymphocyte homing into mesenteric lymph nodes and spleen. In collaboration with another member of the MICMAN consortium, Mikael Skurnik, we experimentally infected the mice and their wild type controls with Yersinia bacteria. Yersinia did not cause any increased mortality among the knockout mice indicating that VAP-1/AOC3 may be safe to block when harmful inflammations are treated. We further confirmed this by blocking the function of VAP-1/AOC3 with combined small molecular inhibitors and function-blocking antibodies and simultaneously induced an experimental bacterial infection into the skin. The skin infection remained equally limited in control treated animals and in mice, which obtained the anti-VAP-1/AOC3 treatment. These results are extremely valuable for the people working with drug development against VAP-1/AOC3.
We have also worked with the development of new antibiotics. This is based on the fact that VAP-1 is an enzyme, semicarbazide sensitive amine oxidase. This enzymatic activity is also present in bacteria. We have found that certain aminoglycoside antibiotics are inhibitors of the enzymatic activity of VAP-1. This knowledge is now used in efforts to develop new antibiotics and specific inhibitors of VAP-1.
Selected publications:
Salmi M, Jalkanen S. Lymphocyte homing to the gut: attraction, adhesion and commitment. Immunol Rev 206:100-13, 2005.
Stolen C, Marttila-Ischihara F, Koskinen K, Yegutkin G, Turja R, Bono P, Skurnik M, Hänninen A, Jalkanen S, Salmi M. Absence of an Endothelial Oxidase AOC3 Leads to Abnormal Leukocyte Traffic in Vivo. Immunity, 22:105-115, 2005
Subproject title: Mucosal immunity in Escherichia coli urinary tract infectionProject leader: Professor Catharina Svanborg, Institute of Laboratory Medicine, Department of Microbiology, Immunology and Glycobiology, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden.Phone: +46 46 173972, E-mail: Catharina.Svanborg@mig.lu.se
Researchers:1G. Bergsten, 1K. Ekström, 1H. Fischer, 1G. Godaly, 1,4L. Gustafsson1M. C. U. Gustafsson, 1L. Hang, 1,5H. Irjala, 3D. Karpman, 1I. Leijonhufvud, 1A-C. Lundstedt, 1B. Ragnarsdottir, 1N. Roche, 1M. Samuelsson, 1P. Samuelsson, 1M. Svensson, 1C. Tallqvist, 1,2B. Wullt,
1Department of Microbiology, Immunology and Glycobiology, Division of Laboratory Medicine, Lund University, 2Department of Urology, Lund university, 3Department of Paediatrics, Lund University, 4Department of Physics, Lund Institute of Technology and Lund University Medical Laser Centre, 5Department of Otorhinolaryngology and Head and Neck Surgery, University of Turku, Turku, Finland
Key words: Urinary tract infection, neutrophil, Toll-like receptor, TLR4, CXCR1, IL-8, asymptomatic bacteriuria, pyelonephritis
Results
1. Identification of genetic defects in patients prone to acute pyelonephritis.
Urinary tract infections (UTIs) are clustered in certain individuals but the molecular basis of disease susceptibility remains unclear. In mice, the susceptibility to UTI is influenced by the murine IL-8 receptor homologue and mutant mice develop acute septic infections and tissue damage due to pathological neutrophil entrapment in the kidneys. This study detected heterozygous, IL-8 receptor (CXCR1) variants in UTI prone children (12/37, 32%) who also expressed less CXCR1 protein and mRNA than age-matched controls (p<0.0001). Sequencing revealed known heterozygous polymorphisms (SNPs) in the intron and exon 2 (24%) and new mutations termed M3-M5 in the 3' untranslated region. The intronic SNP was present in one child without UTI (1/26, 4%) but the exonic SNP and 3' mutations were not detected. Only SNPs 1 and 2 occurred in adult controls, at a lower frequency than in the patients (34/348, 10%, p=0.0134). The intronic SNP1 was shown to reduce the affinity for the transcription factor RUNX1 and the large CXCR1 transcript was reduced in patients carrying the M5 mutation close to the 3' polyadenylation site. Genetic variants of CXCR1 are, thus associated with low CXCR1 expression, which impairs the innate host defense. The results suggest a novel, genetically determined cause of susceptibility to acute pyelonephritis.
2. Discovery of aberrant TLR4 expression in ABU-patients.
TLR4 is essential for the defense against Gram-negative infection, but reduced TLR4 expression has not been linked to altered disease susceptibility in man. This study presents the first evaluation of human TLR4, its adaptors and susceptibility to infection. Patients with UTI were selected for study, as Tlr4 controls the mucosal response to E. coli infection and inactivation of Tlr4 causes an ABU like state (ref). Clearance of infection in Tlr4 wt mice is through neutrophils, but Tlr4 mutant mice have an attenuated response (ref). This study showed that TLR4 expression was lower in children with ABU compared to age-matched controls without UTI. All of the patients expressed detectable amounts of TLR4 on their neutrophils, however, suggesting that the function of TLR4 was attenuated but not lost.
The results suggest that reduced TLR4 expression might protect the urinary tract from the consequences of inflammation, including symptoms and tissue damage. Individuals who have stably reduced TLR4 expression might run a reduced risk of developing symptomatic UTI, due to the poor inflammatory response. Consistent with this hypothesis, the children with primary ABU had the lowest TLR4 levels. In addition, low TLR4 expression might be an essential mechanism to avoid frequent responses to recurrent bacterial challenge in UTI susceptible individuals. The patients with secondary ABU had experienced prior symptomatic infections, suggesting that their TLR4 expression had been intact and that they were able to respond to the uropathogenic E.coli strains that activate TLR4 signaling through fimbriae and LPS. The mechanism of reduced TLR4 expression might thus differ between these patient groups.
3. Bone marrow transplantation in mIL-8Rh KO mice.
CXCR2 -/- mice are susceptible to experimental urinary tract infection (UTI) due to deficient neutrophil migration and bacterial elimination. Following mucosal infection, neutrophils accumulate under the pelvic epithelium and if the mice do not succumb to acute septic infection, the frustrated neutrophil response causes renal scarring. CXCR2 is expressed on neutrophils and epithelial cells, and is involved in neutrophil exit across the epithelial barrier and phagocytosis.
The relative importance of CXCR2 expression on neutrophils and epithelial cells was investigated using bone marrow transplantation. CXCR2 -/- mice and congenic Balbc mice were lethally irradiated and transplanted with bone marrow from CXCR2 -/- or Balbc mice, respectively. After 8 weeks, the mice were exposed to UTI, using an E. coli pyelonephritis strain.
Mice receiving CXCR2 -/- marrow showed high spontaneous mortality due to sepsis (80% in CXCR2 -/- to CXCR2 -/- and 65 % in Balbc receving CXCR2 -/- marrow), which decreased following antibiotic treatment. Balbc bone marrow protected 95% of the CXCR2 -/- mice. In the Balb/c background, 80% of transplanted mice survived without antibitotics. The intestinal flora was a likely source of infection.
Surviving mice were subjected to experimental UTI and the mucosal neutrophil response was recorded. Balb/c mice receiving CXCR2 -/- bone marrow had a significantly lower neutrophil response (p=0.048) than Balb/c receiving autologous bone marrow. The neutrophil response in CXCR2 -/- mice was reconstituted by Balbc bone marrow to a level similar to Balbc mice with autologous bone marrow. CXCR2 -/- reciving autologous bone marrow had no neutrophil response. Kidney sections showed difference in expression of CXCR2, MIP-2 and neutrophil marker RB6-8C5.
Bone marrow transplantation was used to evaluate the relative importance of epithelial and neutrophil CXCR2 expression for neutrophil exit across the mucosa. We conclude that an intact neutrophil CXCR2 expression is more important than the uroepithelial receptor expression.
Subproject: Recruitment of innate cell populations during Salmonella infection
Project leader: Professor Mary Jo Wick,Dept. Medical Microbiology and Immunology, Göteborg University, Guldhedsgatan 10 143 46 Göteborg, tel. 031 342 4602, mary-jo.wick@immuno.gu.se
Researcher: Anna Rydström, Ph.D. student
Results
Salmonella typhimurium is a Gram-negative intracellular bacterium causing an infection in mice that resembles human typhoid fever. The mesenteric lymph nodes (MLN) and Peyer's patches (PP) are the first organs encountered by orally acquired Salmonella. These lymphoid organs are entry points of the bacteria as well as targets of the infection. C57BL/6 mice were infected orally with Salmonella and the population dynamics of innate cells in MLN and PP were examined between days 2 and 5 post infection.
Cells from MLN and PP in naïve and mice orally infected 2, 3 or 5 days earlier with Salmonella were analyzed by flow cytometry. Recruited monocytes and neutrophils were defined as non-overlapping populations based on the expression of CD68 and Gr-1. Monocytes were identified as CD68hiGr-1int cells and eutrophils as CD68low Gr-1hi cells.
Flow cytometry analysis on cells prepared from the PP and MLN of naïve and Salmonella-infected mice revealed that both monocytes and neutrophils were recruited to the MLN and PP of Salmonella-infected mice. In contrast, these cell populations were nearly absent in the same tissues of naïve mice. The recruited monocyte population compromised 3% of total cells in MLN and PP at day 5 post infection. Further characterization of the monocyte population in infected mice showed that the cells express low levels of CD62L, an intermediate to high level of MHC-II and an intermediate level of CD86 and CD80. They also express a low level of CD11c. This latter observation excludes the possibility that they are conventional dendritic cells, which express very high levels of CD11c. Indeed, direct comparison of the phenotype and activation state of dendritic cells and recruited monocytes in the same infected individual revealed distinct differences. The monocytes but not dendritic cells of infected animals produced anti-bacterial effector molecules including TNF-a and iNOS. These effector functions became apparent when the monocytes arrived in infected tissues, as the same cells were present in the blood of infected mice but did not exhibit effector functions.
The absolute number and percentage of other immune cell populations were also determined by flow cytometry analysis of cells from MLN and PP in infected compared to naïve individuals. Natural killer (NK) cells were defined as NK1.1+TCRab- cells while B cells were identified as B220+ and T cells as TCRab+NK1.1- cells. The number of NK1.1+ NK cells was slowly reduced during infection while the B cell number doubled in MLN. In PP, the percentage of B and T cells remained relatively stable at day 2 but showed a small drop at day 5 post infection.
Conclusions
Monocytes and neutrophils are rapidly recruited to MLN and PP early during Salmonella infection, and the monocytes are the main cells producing anti-bacterial effector molecules.
The recruited CD68hiGr-1int monocytes outnumber Gr-1hi neutrophils and conventional dencritic cells in the MLN and PP of infected mice. The recruited moncoytes express an intermediate to high level of MHC-II, an intermediate level of CD86, CD80,CD11c and a low level of CD62L.
Blood monocytes have a similar phenotype to the tissue monocytes in infected mice, but effector functions are not apparent until the cells arrive in the tissues.
The accumulation of innate cells correlates with increased bacterial load during the infection.
The number of NK cells go down in MLN of B cells double at day 5 post infection. In PP, the percentage of both NK1.1+ cells and B cells decreases slightly.
Selected publication
Rydström, A. and Wick, M.J. (2006). Monocyte recruitment to the gut-associated lymphoid tissue in mice orally infected with Salmonella. J. Immunol. Submitted.
An abstract of the research proposal (January 2003)