The role and regulation of the BH3‐only protein Bim in immune suppression during sepsis

Submission note: A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry and Genetics, Institute for Molecular Science, College of Science, Health and Engineering, La Trobe University, Bundoora.

Sepsis is a complex inflammatory disorder that leads to substantial mortality in non‐coronary ICUs. Sepsis incidence after surgery trebled in the last 10 years and are estimated to further increase due to an aging population and comorbidities. Improved protocols for the treatment of acute sepsis have resulted in most patients surviving the early hyper‐inflammatory stage and entering a protracted immune suppressive phase, which accounts for most of the fatalities. This phase is characterized by extensive apoptosis in the cells of the adaptive immune system leading to prolonged lymphocytopenia. This makes the patients susceptible to nosocomial infections and is now considered an independent predictor of patient survival in sepsis. Independent reports suggested systemic circulating immunomodulatory factors in sepsis patients that mediate cellular apoptosis and immune suppression. The BH3‐only protein Bim was previously shown to be a major player in sepsis‐mediated immune cell apoptosis, however the molecular mechanisms remain elusive. In the present study, a central role for Bim during sepsis was confirmed both in vivo and in a newly established in vitro sepsis model assay system. The induction of endoplasmic reticulum stress was discovered to be the prevalent mechanism of Bim induction and blockage of ER stress led to improved lymphocyte survival. Biochemical purification identified a secreted form of the ER chaperone BiP/Grp78 as the apoptosis‐inducing factor. BiP was previously described to have immunomodulatory functions and therefore presents an exciting novel therapeutic target for sepsis. In the present study, monoclonal antibodies against recombinant human BiP were generated and validated in vitro as therapeutic tool for treatment of sepsis‐associated lymphocytopenia. Furthermore, a diagnostic ELISA was established and could show elevated levels of secreted BiP in septic mice and human sepsis patients and therefore BiP has the potential to be used as novel sepsis biomarker.