In vivo imaging of the regulatory mechanisms in acute and chronic delayed type hypersensitivity reactions

Abstract

Inflammatory responses are involving a complex interplay between molecular and cellular mechanisms. Here in this thesis, different players within the inflammatory tissue and draining lymph nodes such as cathepsin B, reactive oxygen species (ROS) and reactive nitrogen species (RNS) as well as the NF κB pathway were investigated in a model of acute and chronic delayed type hypersensitivity reaction (DTHR) by non invasive in vivo optical imaging. Cathepsin B represents an important intra- and extracellular protease, which is of immense importance during the establishment of an immune response e.g. in antigen processing. ROS and RNS are a byproduct of cellular metabolism but are also produced as effector and signaling molecule mainly by neutrophilic granulocytes. NF-κB is an important signaling pathway which is sensing inflammatory stimuli and leads to the expression of many genes involved in the immune response. The T-cell driven acute cutaneous DTHR was induced by sensitizing mice at the abdomen and eliciting the inflammatory response 7 days later at the right ear using the hapten 2,4,6-trinitrochlorobenzene (TNCB). Repetitive application of TNCB at the right ear for up to five times induced chronic DTHR. Beside wild-type C57BL/6 mice, we used cathepsin B-deficient (Ctsb-/- ), cathepsin Z-deficient (Ctsz-/-) and NF-κB-luciferase-reporter mice to induce the acute and chronic TNCB specific DTHR. Cathepsin B activity, ROS/RNS production and NF-κB activation were measured noninvasively by optical imaging employing protease-activatable fluorescence probes, the ROS-sensitive chemiluminescence probe L-012 and luciferin for bioluminescence imaging in NF-κB-luciferase-reporter mice. Extensive ex vivo validation was performed including histopathology, immunohistochemistry, flow cytometry, fluorescence microscopy, RT-PCR as well as active site labeling of proteases and Western blotting. Furthermore, the therapeutic effects of N-acetylcysteine (NAC) and the cathepsin-inhibitors CA-074 and Inhibitor 17 were investigated. In acute cutaneous DTHR in vivo optical imaging detected an intense cathepsin B activity as well as ROS/RNS production and NF-κB activation peaking at 24 h after the 1st TNCB ear challenge. In chronic DTHR the cathepsin B activity further increased while the peaks of ROS/RNS production and NF-κB activation were shifted to an earlier timepoint. NAC treatment decreased the ear swelling response in acute and chronic DTHR while the influence on the ROS/RNS production and NF-κB activation during acute and chronic DTHR assessed by in vivo optical imaging was divergent. The cathepsin B inhibitors CA-074 and Inhibitor 17 reduced inflammation in acute but not in chronic DTHR, while Ctsb-/- mice exhibited an even enhanced ear swelling response during acute DTHR caused by a compensatory expression of cathepsin Z. Ex vivo analysis revealed enhanced cathepsin B expression in neutrophilic granulocytes, dendritic cells, macrophages, B, T and natural killer (NK) cells within inflamed ears and draining lymph nodes. The investigated mechanisms are an essential part of the multifaceted interplay which is needed to establish and maintain inflammatory immune responses. Molecular imaging involving optical imaging is a highly capable tool to monitor these mechanisms in vivo and to asses targeted therapeutic interventions. These results could be of high importance not only to modulate inflammatory autoimmune diseases like rheumatoid arthritis but also to prevent carcinogenesis in chronic inflammation like non alcoholic steatohepatitis.