Severe sepsis still represents a major challenge in medicine. Epidemiologic studies indicate that severe sepsis causes as many deaths annually as acute myocardial infarction. However, the under¬standing of the immune pathology of sepsis is still incomplete and adjuvant immune interventions (blockade of bacterial components [LPS] or host cytokines [TNF, IL-1]) have not been successful so far. Although the reasons for this failure are complex and multifactorial, the use of clinically inap¬propri¬ate animal models and the focus on single effector molecules may represent major problems [1]. Thus, previous concepts for the immune pathology of sepsis were mainly based on animal models reflecting intoxication of the host rather than infection (i.e. administration of bacterial toxins or high doses of live bacteria) and analysis of the host response pattern to a septic challenge was limited to the regulation and function of a single or a few genes.
To circumwent these problems, novel functional genomics methodologies including microarrays and bioinformatic approaches were applied to idenify global response patterns to septic conditions using a clinically relevant model of septic peritonitis (CASP) or to systemic bacterial infection with Listeria monocytogenes [2, 3]. Specifi¬cally, we have established key transcriptional maps for various tissues and organs from mice subjected to these septic challenges. In the CASP model, we have shown that the common Toll-like receptor (TLR) adapter protein MyD88 controls a signaling pathway that leads to hyperinflam¬mation, organ injury, and increased lethality. Using microarray-based expression profiling we have identi¬fied a specific gene set regulated by the MyD88 pathway in sepsis. Importantly, a previously unappreciated cell type-specific function of TLR signaling pathways mediated by the adapter proteins MyD88 and TRIF was identified. Parallel studies in surgical patients revealed TLR4-medi¬ated response patterns that are associated with sepsis occurence and outcome.
Combining these achievements with conditional gene inactivation technology in future work it will be possible to address the individiual role of cellular subsets, immune receptors and signaling components for the diagnosis and therapy of sepsis.
Results
The potential role of the common TLR signaling protein MyD88 in polymicrobial septic peritonitis was investigated using the mouse CASP model. Whereas bacterial clearance was normal in MyD88-deficient mice, the systemic inflammatory response was strongly attenu¬ated in the absence of MyD88 [4]. Surprisingly, microarray analyses revealed that MyD88-deficiency did not alter cytokine and chemokine production in spleen, but markedly reduced the inflammatory response in liver and lung. These results were confirmed by protein expression analysis (submitted for publication). These results imply a central role of MyD88 for the systemic immune pathology of polymicrobial sepsis and show that the cytokine response is regulated in an organ-specific manner.
Differential regulation of TLR signaling was further examined in vitro. Microarray ex¬pression profiling of dendritic cells identified TRIF as a major regulator of the TLR4-triggered activation program [5]. The results suggested that TLR4-promoted Th1 responses are TRIF-dependent and indicated that TRIF regulates TLR4-mediated gene expression both by type I IFN-dependent and -independent mecha¬nisms. Functional TRIF was also required for the nor¬mal induction of numerous genes considered important for host defense against diverse patho¬gens. Importantly, the contribution of TRIF and MyD88 to TLR4-mediated gene in¬duction differed between dendritic cells and macro¬phages thereby demonstrating a previ¬ously unappreciated cell type-specific function of adapter proteins for TLR signaling.
Complete transcriptome maps were established from macrophages after TNF, IFN# and TGF# stimulation and differentially regulated genes (approx. 25) are currently thoroughly analysed for regulation in tissues of mice that underwent CASP surgery or listeria infection. Furthermore, transcriptome profiling has been performed for liver and spleens of wildtype and TNFRp55- deficient mice for the elucidation of key response genes allowing survival of the host after infection with intracellular bacteria which will be systematically gene targeted. One novel gene identified using such a screening approach was PUMA-G. A PUMA-G deficient mouse line has been established by us already. Phenotypic analysis revealed an impaired immunological host response [6, 7].
To determine the relevance of the findings obtained in preclinical mouse models a patient study was performed. The aim of this study was to identify TLR4-mediated response patterns of peripheral monocytes that would be predictive for the occurrence or outcome of postoperative sepsis. The results showed that IL-12 production was significantly higher in patients surviving a postoperative sepsis as compared with patients with lethal sepsis. In multivariate analysis, IL-12 was the only factor associated with lethal outcome of postoperative sepsis. When patients were further analyzed according to underlying disease and treatment, preoperative IL 12 production was only associated with prognosis in patients receiving neoadjuvant radiochemo¬therapy (submitted for publication).
Outlook
The major aim of this project is to understand how cell-type specific TLR signaling deter¬mines the immune response to sepsis. We propose to express MyD88 and TRIF, which regulate the major signaling pathways of TLRs, in a cell-type specific manner in mice using a "switch-on" strate¬gy of conditional gene targeting that was established in our previous work. Expression of MyD88 and TRIF will be selectively induced in phagocytes, macrophages, dendritic cells, or endothelial cells.
Mutant mice will be investigated in the CASP model of polymicrobial septic peritonitis and in a model of septic listeriosis. The analysis of mutant mice includes regulation of the innate immune response at the transcriptome level as well as determination of pathogen defence and organ injury. Together with the transcriptome data obtained from conventional knockout models and surgical sepsis patients during NGFN1 the new data will be subjected to an integrated analysis of gene expression profiles aiming at the identification of key regulatory pathways and gene sets that determine sepsis outcome.
Mutant mouse strains will be exchanged with other subprojects of NGFN2 to identify the role of cell type-specific TLR signaling for the immune defence against various pathogens. In turn, expertise in mouse sepsis models will be made available to other research groups in NGFN2.
In addition to the generation and analysis of conditional mutant mouse strains, cell type-specific aspects of TLR signaling will be analyzed by biochemical methods. To this end, in vitro differentiated macrophages and dendritic cells will be analyzed for differences in the activation of MAP kinases as well as NF-#B and IRF-3 transcription factors.
Lit.: 1. Buras J et al. Animal models of sepsis: setting the stage. Nat Rev Drug Discov. 2005; in press. 2. Maier S et al. Cecal ligation and puncture versus colon ascendens stent peritonitis: two distinct animal models for polymicrobial sepsis. Shock. 2004; 21:505-12. 3. Ehlers S, et al. The lymphotoxin beta receptor is critically involved in controlling infections with the intracellular pathogens Mycobacterium tuberculosis and Listeria monocytogenes. J Immunol. 2003; 170:5210-8. 4. Weighardt H, et al. Cutting edge: MyD88 deficiency improves resistance against sepsis caused by polymicrobial infection. J Immunol. 2002; 169:2823. 5. Weighardt H, et al. Identification of a TLR4- and TRIF-dependent activation program of dendritic cells. Eur J Immunol. 2004; 34:558-64. 6. Schaub A et al. PUMA-G, an IFN-#-inducible gene in macrophages is a novel member of the seven transmembrane spanning receptor family. Eur J Immunol. 2001; 31:3714-25. 7. Tunaru, S et al. PUMA-G/HM74 is a receptor for nicotinic acid and mediates its antilipolytic effect. Nat Med. 2003; 9:352-5.
