New therapeutic approach for intra-abdominal abscess, peritonitis and sepsis: Liposome mediated gene transfer of the anti-inflammatory cytokines Interleukin-4, Interleukin-10 and Interleukin-13

Septic Shock is a clinical syndrome that warrants increasing efforts to find live-saving pharmacologic interventions. Numerous studies aimed at the downregulation of the overwhelming production of pro- inflammatory cytokines (TNF-alpha, Interleukin-1) during sepsis. However, the use of monoclonal antibodies directed against TNF-alpha, the blockade of the receptors of TNF-alpha and Interleukin-1 as well as the effect of the potent anti-inflammatory cytokine Interleukin-10 fell short of expectations. Thus, systemic administration of cytokine inhibitors does not seem to be an efficient treatment modality for reducing the overwhelming production of pro-inflammatory cytokines in the course of sepsis. This might be mainly due to the fact that systemic administration of cytokine inhibitors at levels sufficient to neutralize exaggerated TNF-alpha production in one tissue compartment may also block the presumably beneficial aspects of cytokine production in other tissue compartments. Gene transfer seemed to us the most promising modality to circumvent the disadvantages of a systemic administration of pro-inflammatory cytokine inhibitors in septic shock. In our previous studies we were able do demonstrate that the local delivery of the anti-inflammatory cytokines Interleukin-4, Interleukin-10 and Interleukin-13 by liposome mediated gene transfer did not only improve survival in a murine LPS-shock model but also restored the immuncompetence of the peritoneal macrophages. So far, our studies concentrated on the hyperinflammatory phase of sepsis, the lethal septic shock. The goal of our future studies to be reached is the preventive as well as the therapeutic treatment of the late chronic phase of septic patients. Animals made LPS tolerant and their LPS-desensitized cells will serve as an experimental model for the state of the diminished host defense. The demonstrated potent modulating effects of Interleukin-4, Interleukin-10 and Interleukin-13 in our lethal endotoxin shock model warrant the conviction that these three anti-inflammatory cytokines can also restore the TNF-alpha response to endotoxin in LPS-desensitized mice. Subsequently, we will direct our attention to the treatment of intra-abdominal abscesses and peritonitis. Cecal ligation and puncture offers us a well established lethal and non-lethal murine model for peritonitis. Since we strongly believe in the local compartmentalized intervention and treatment of peritonits and intra-abdominal abscesses, we plan to investigate the potent modulatory effects of liposome mediated gene transfer of Interleukin-4, Interleukin-10 and Interleukin-13 in both models, concerning survival of the animals and immunocompetence of macrophages. Once these studies are completed, effort will be made to document whether the liposom mediated gene therapy alters nonspecific immune functions, and predisposes these animals to increased morbidity from infections. To address this latter point, we will employ a Listeriosis model. We believe that by the means of all these studies we will get nearer to our final goal, the life-saving treatment of chronic septic patients.

Lipopolysaccharide (LPS) has been proven to be the prime initiator of gram-negative bacterial sepsis resulting in an overwhelming production of pro-inflammatory cytokines such as TNF-a , Interleukin (IL)-1, IL-6, IL-12 and anti-inflammatory cytokines e.g. IL-10. However, the precise mechanism, responsible for the activation of cells by LPS, has been a long standing enigma. Recent data identified the Toll-like receptor 4 (TLR4) as the predominant LPS receptor, mediating LPS induced cellular activation. Toll was originally discovered on the basis of its role in establishing the dorsoventral axis in the early Drosophila embryo, but further studies asigned an additional important role to the Toll-family members in antifungal and antibacterial immune response in Drosophila. In mammals, Toll-like receptors were shown to function as profound pattern recognition receptors (PRRs) of the innate immune system. In our study, we focused on the hypoinflammatory phase of sepsis, characterized by an overall refractoriness of the host to re-exposure to LPS. Monocytes from these late-stage septic patients produce diminished amounts of TNF-a , IL-1, IL-6, IL-8 and IL- 12 when stimulated ex vivo with LPS. Our main goal was to overcome this immunoparalysis, a phenomenon known as endotoxin tolerance, to reduce the risk of the late-stage septic patient to die from secondary infections. We employed local liposome mediated gene transfer of the cytokines IL-10 and IL-13 in a non-lethal murine endotoxemia model. Balb/C mice were experimentally rendered refractory to LPS and at the same time transfected with hIL-10 or hIL-13 plasmid DNA complexed to liposomes. Peritoneal macrophages were then stimulated in vitro with LPS or PMA and TNF-a and IL-12 production was evaluated by ELISA. Unexpectedly, our data demonstrated that local liposome mediated gene transfer of hIL-10 and hIL-13 could specifically restore TNF-a production by peritoneal macrophages upon restimulation with LPS, therby breaking LPS tolerance. IL-12 production could not be rescued by any of the two cytokines used. In order to evaluate the molecular background of this surprising but promising result, we planned to investigate the role of the LPS receptor TLR4 in the abolishment of LPS tolerance by gene transfer of IL-10 and IL-13 in this non-lethal, murine endotoxemia model. We tried and are still trying to generate a monoclonal antibody against muTLR4 in the rat. Recombinant muTLR4 expressed in E.coli was the antigen of choice for immunization. Due to the formation of inclusion bodies and insolubility of recombinant muTLR4 even under denaturing purification conditions, we decided to use live, transfected cells expressing high amounts of muTLR4 as immunogen. Despite a measurable titre of specific antibodies against muTLR4 in the serum and more than 900 hybridoma clones in the resulting fusions, we could not detect any specific mAb against muTLR4. To overcome this technical problem in the future, different expression systems for recombinant muTLR4 protein, including yeast and insect cells, as well as naked DNA will be used to generate a monoclonal Ab against muTLR4.