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Interaction of bacterial pathogens with innate immune cells
Innate immune cells detect invading bacterial pathogens by the recognition of pathogen-associated molecular patterns (PAMPs) by Toll-like (TLR), NOD-like (NLR) and RIG-like receptors (RLRs). While TLRs and RLRs induce the production of proinflammatory cytokines and type I interferons, NLRs like NLRPs control the release of mature IL-1b via activation of caspase 1. We study the interaction of four different bacterial pathogens with innate immune cells in vivo and in vitro: Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia muridarum and uropathogenic Escherichia coli. Aside from these projects, we develop molecules which impede the activity of nucleotide-binding TLRs or which bind to MyD88, the central adaptor of the TLR-signaling cascade.
A) Interaction of Chlamydia with Toll-like receptors
Chlamydia are obligate intracellular bacteria with an unique replication cycle. Elementary bodies of Chlamydia are metabolically inert, but are able to infect cells. Inside cells elementary bodies develop within a vacuole to reticular bodies, which are metabolically active and multiply. Subsequently, reticular bodies revert back to elementary bodies which leave the host cell to infect neighboring cells. The intracellular bacteria are able to survive within the host cell since they prevent the fusion of lysosomes with the bacteria-containing vacuole. C. trachomatis causes ocular trachoma, inclusion conjunctivitis and urogenital infections. C. pneumoniae is wide-spread within the population since up to 70% of healthy individuals are antibody-positive. Clinically, C. pneumoniae is responsible for a variety of diseases like atypical pneumonia, sinusitis and bronchitis. Furthermore, the organism was implicated in the pathogenesis of atherosclerosis, a conclusion which is heavily debated. C. muridarum (also called C. trachomatis mouse pneumonitis strain) causes genital tract infections and pneumonia in mice.
Our own studies focused on the host-parasite relationship of this obligate intracellular bacterium with cells of the innate immune system. We demonstrated that bone marrow-derived dendritic cells were rapidly activated upon infection with C. pneumoniae. The cells recognized the microorganism via TLR2 and TLR4, up-regulated the expression of MHC class II molecules and co-stimulatory molecules like CD40, CD80, and CD86 and released pro-inflammatory cytokines like TNF-a and IL-12p40.
To define the chlamydial TLR-ligand we were able to show that purified chlamydial endotoxin from C. trachomatis stimulates TNF-secretion in a TLR4-dependent manner. However, the whole microorganism C. trachomatis activated innate immune cells independent from TLR4. Therefore, we concluded that other chlamydial TLR-ligands were recognized during infection.
Another potential PAMP of Chlamydia is their heat shock protein 60 (HSP60). Exploration of the stimulatory capacity of purified HSP60 from C. pneumoniae for dendritic cells revealed that the protein stimulated these cells in a TLR2 and TLR4-dependent fashion. In vivo, purified HSP60 was able to induce the secretion of chemokines like KC and to recruit neutrophils upon intra-peritoneal injection. Both host responses were again dependent on TLR2 and TLR4. Unlike LPS, purified HSP60 failed to induce TNF-secretion. Thus, based on the cytokine profile induced, HSP60 stimulated innate immune cells in a way which differed from LPS.
TLR2 and TLR4 as well as the adapter molecule MyD88 were also crucial to detect C. pneumoniae in vivo, since mice double-deficient for TLR2 and TLR4 or deficient for MyD88 were impaired to secrete chemo- and cytokines upon pulmonary infection, failed to recruit polymorphonuclear neutrophils (PMN), were unable to control chlamydial burden during the later stage of the disease and finally succumbed to the infection. Using microarray technology we also reported that more than 200 genes were not or much weaker induced in infected MyD88-deficient mice compared to wild type mice.
A surprise came with our discovery that PMNs were able to increase chlamydial replication in vivo and in vitro. This surprising result explained why MyD88-deficient mice unexpectedly displayed a lower chlamydial burden compared to wild type mice at the beginning of the infection although the microorganism was not detected: MyD88-deficient mice were unable to recruit PMNs.
Further exploration of the molecular basis for PMN-mediated enforcement of chlamydial replication revealed that a soluble factor of PMNs was responsible for this phenomenon. This soluble factor was later identified as IL-6. The mechanism, how IL-6 influences chlamydial growth, is unknown and requires further investigations.
T-cell-derived IFN-gamma is important to control a chlamydial infection. We showed that TLR2 and TLR4 negatively regulate the generation of IFN-gamma-producing T-cells. Thus, TLR2/4 double-deficient mice develop higher IFN-gamma levels than wild type mice post pulmonary infection with C. pneumoniae, since more IFN-gamma-secreting T-cells were found in the lung and spleen of TLR2/4-deficient mice. This finding correlated with a reduced number of regulatory T-cells present in the infected lung of TLR2/4 double-deficient mice. Taken together, since TLR2/4 double-deficient mice recruited fewer Tregs into the lung, chlamydia-specific T-cells could produce higher IFN-gamma levels in these mice.
Current and future investigations:
Females are often infected with C. trachomatis without overt signs of disease. Nevertheless, the infection leads to salpingitis which may lead to infertility. To prevent this process a vaccine against C. trachomatis is urgently required. Since a successful vaccine should prevent disease sequela we established a murine genital tract infection model with C. muridarum which leads to hydrosalpinx in mice several weeks post vaginal infection. Using different chlamydial proteins, which are either secreted or expressed in the cell wall or in the inclusion membrane, in combination with CpG-DNA as adjuvants we hope to prevent the pathologic consequences of this infection.
1. Prebeck, S., C. Kirschning, S. Dürr, C. da Costa, B. Donath, K. Brand, V. Redecke, H. Wagner and T. Miethke. Predominant role of toll-like receptor 2 versus 4 in Chlamydia pneumoniae-induced activation of dendritic cells. 2001. J. Immunol. 167: 3316-23
2. Prazeres da Costa, C., C.J. Kirschning, D. Busch, S. Dürr, L. Jennen, U. Heinzmann, S. Prebeck, H. Wagner and T. Miethke. Role of chlamydial heat shock protein 60 in the stimulation of innate immune cells by Chlamydia pneumoniae. 2002. Eur. J. Immunol. 32: 2460-70
3. Prazeres da Costa, C., F.-J. Neumann, A. Kastrati. I. Stallforth, M. Schmid, N. Jogetai, S. Prebeck, H. Wagner and T. Miethke. Role of IgG-seropositivity in early thrombotic events after coronary stent placement. 2003. Atherosclerosis. 166: 171-6
4. Prebeck, S., Brade, H., Kirschning, C.J., Prazeres da Costa, C., Dürr, S., Wagner, H. and T. Miethke. The gram-negative bacterium Chlamydia trachomatis L2 stimulates TNF-secretion independently of its endotoxin. 2003. Microbes Inf. 5: 463-70.
5. Da Costa CU, N. Wantia, C.J. Kirschning, D.H. Busch, N. Rodriguez, H. Wagner and T. Miethke. Heat shock protein 60 from Chlamydia pneumoniae elicits an unusual set of inflammatory responses via Toll-like receptor 2 and 4 in vivo. 2004. Eur. J. Immunol. 34:2874-84.
6. Rodriguez N., F. Fend, L. Jennen, M. Schiemann, N. Wantia, C. U. Prazeres da Costa, S. Dürr, U. Heinzmann, H. Wagner and T. Miethke. Polymorphonuclear neutrophils improve replication of Chlamydia pneumoniae in vivo upon MyD88-dependent attraction. 2005. J. Immunol. 174:4836-4844.
7. Rodriguez N., N. Wantia, F. Fend, S. Durr, H. Wagner and T. Miethke. Differential involvement of TLR2 and TLR4 in host survival during pulmonary infection with Chlamydia pneumoniae. 2006. Eur. J. Immunol. 36:1145-1155.
8. Rodriguez N., J. Mages, H. Dietrich, N. Wantia, H. Wagner, R. Lang and T. Miethke. MyD88-dependent changes in the pulmonary transcriptome after infection with Chlamydia pneumoniae. 2007. Physiol Genomics. 30:134-45.
9. Rodriguez, N., R. Lang, N. Wantia, C. Cirl, T. Ertl, S. Dürr, H. Wagner and T. Miethke. Induction of iNOS by Chlamydophila pneumoniae requires MyD88-dependent activation of JNK. 2008. J. Leukoc. Biol. 84:1585-93.
10. Peschel, G., L. Kernschmidt, C. Cirl, N. Wantia, T. Ertl, S. Dürr, H. Wagner, T. Miethke and N. Rodríguez. Chlamydophila pneumoniae downregulates MHC-class II expression by two cell type-specific mechanisms. 2010. Mol. Microbiol. 76:648-61. TM and NR share senior authorship.
11. Rodriguez N., H. Dietrich, I. Mossbrugger, G. Weintz, J. Scheller, M. Hammer, L. Quintanilla-Martinez, S. Rose-John, T. Miethke and R. Lang. Increased inflammation and impaired resistance to Chlamydophila pneumoniae infection in Dusp1(-/-) mice: critical role of IL-6. 2010. J. Leukoc. Biol. 88:579-87. TM and RL share senior authorship.
12. Wantia N., N. Rodriguez, C. Cirl, T. Ertl, S. Dürr, L.E. Layland, H. Wagner and T. Miethke. Toll-like receptors 2 and 4 regulate the frequency of IFNγ-producing CD4+ T-cells during pulmonary infection with Chlamydia pneumoniae. 2011. PLoS One. 6:e26101.
B) Bacterial Toll/Interleukin-1 resistance proteins: analysis of their function
A number of human bacterial pathogens like uropathogenic E. coli strains, Brucella spp. and the S. aureus strain MSSA476 possess genes which encode for proteins containing a Toll/Interleukin 1 receptor (TIR) domain. Based on their aminoacid sequence and their predicted tertiary structure we hypothesized that these proteins might interfere with TLR-signaling.
FlAsH-tagged TcpC (green) in the cytosol of macrophages
Our published data show, that the TIR-containing protein from the E. coli strain CFT073 (TcpC) and from B. melitensis (TcpB) lowered the secretion of pro-inflammatory cytokines like TNF or IL-6. Both proteins bound to the adapter molecule MyD88 and impaired signaling of TLRs like TLR2, TLR4, TLR5, TLR9, but not TLR3. CFT073 benefited from TcpC in that the bacteria accumulated intracellularly to higher numbers than a tcpC-deficient strain. TcpC was secreted by CFT073 and the protein was subsequently taken up by host cells into the cytosol. Based on these findings we added TcpC to the culture supernatant of innate immune cells and also switched off TLR-signaling. The potency of this new virulence factor was explored in vivo. Compared to the tcpC-deficient strain we observed that the bacterial burden of CFT073 in the urine and kidneys was 100-1000 fold higher. Furthermore, CFT073 caused kidney abscesses while this complication was not detected with the tcpC-deficient strain. The secretion of TcpC by CFT073 could be prevented by the efflux pump inhibitor PAbN. This result provides the basis of a new treatment strategy which inactivates this potent new virulence factor and may be applied in addition to antibiotics.
In a close collaboration with T. Xiao et al. TcpC was found to be crucial to crystalize MyD88. Although we obtained no co-crystal of MyD88 with TcpC, the crystal structure of the MyD88 TIR domain revealed distinct loop conformations which differ from corresponding TLR-loops and underscore the functional specialization of the adapter, receptor, and microbial TIR domains. Our structural analyses shed light on the genetic mutations at these loops. We also demonstrated that TcpC directly associated with MyD88 and TLR4 through its predicted DD and BB loops to impair the TLR-induced cytokine induction. Furthermore, NMR titration experiments identify the unique CD, DE, and EE loops from MyD88 at the TcpC-interacting surface, suggesting that TcpC specifically engages these MyD88 structural elements for immune suppression. These findings thus provided a molecular basis for the subversion of TLR signaling by the uropathogenic E. coli virulence factor TcpC and furnished a framework for the design of novel therapeutic agents that modulate immune activation.
Current and future investigations:
We presently investigate, whether other recognition systems of innate immune cells are influenced by Tcps.
Furthermore, we intend to define the bacterial secretion and eukaryotic uptake mechanisms which allow TcpC to move from the bacterium to the cytosol of host cells.
1. Cirl C., A. Wieser, M. Yadav, S. Duerr, S. Schubert, H. Fischer, D. Stappert, N. Wantia, N. Rodriguez, H. Wagner, C. Svanborg and T. Miethke. Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins. 2008. Nat Med. 14:399-406.
2. Cirl, C. and T. Miethke. Microbial Toll/interleukin 1 receptor proteins: a new class of virulence factors. 2010. Int . J. Med. Microbiol. 300:396-401.
3. Yadav, M., J. Zhang, H. Fischer, W. Huang, N. Lutay, C. Cirl, J. Lum, T. Miethke and Svanborg C. Inhibition of TIR domain signaling by TcpC: MyD88-dependent and independent effects on Escherichia coli virulence. 2010. PLoS Pathog. 6:e1001120.
4. Snyder G.A., C. Cirl, J. Jiang, K. Chen A. Waldhuber, P. Smith, F. Römmler, N. Snyder, T. Fresquez, S. Dürr, N. Tjandra, T. Miethke, T.S. Xiao. Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic Escherichia coli. 2013. Proc Natl Acad Sci U S A. 110:6985-90. TM and TX are corresponding authors.
C) Development of molecules which impair the TLR-system
Based on our findings of the TcpC-MyD88 interface we defined peptides derived from certain regions of TcpC to inhibit TLR-signaling. These peptides bound to MyD88 or TLR4 and impaired TLR-signaling. To further improve their inhibitory potential, the amino-acid sequence of the peptides needs to be optimized.
Similarly, we tested in close cooperation with M. Jurk, E. Uhlmann and J. Vollmer a series of inhitibitory oligonucleotides for their potential to impair TLR3, TLR7 and TLR9-induced responses of a variety of murine innate immune cells. We found that certain modifications of these inhibitory oligonucleotides improved their TLR-inhibitory function significantly. Future work will unravel whether human immune cells can be inhibited with similar effectiveness.
1. Guanine-modification of inhibitory oligonucleotides potentiates their suppressive function. Römmler, F., M. Jurk, E. Uhlmann, M. Hammel, A. Waldhuber, L. Pfeiffer, H. Wagner, J. Vollmer, and T. Miethke. 2013. In press
Univ. Prof. Dr. med. Thomas Miethke
Direktor des Instituts