Mechanistically defining the role for each NFkB family member in pro-inflammatory macrophage activation

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PROJECT SUMMARY The World Health Organization (WHO) estimates that ~6 million people die from sepsis each year, with the greatest burden felt in low to middle income countries. According to the WHO, one out of ten deaths during pregnancy is a result of maternal sepsis with over 95% of these cases arising in developing countries. Finally, the Centers for Disease Control and Prevention, estimates that 3-17% of all patients with COVID-19 will develop acute respiratory distress syndrome (ARDS), the primary cause of mortality in this patient population. Sepsis and ARDS, represent severe consequences of immune system overactivation and dysregulation during infection. Although the burden of this pathology is recognized, the molecular underpinnings of this clinical deterioration remain poorly understood. NFkB is a ubiquitous transcription factor that is activated during viral and bacterial infection to control innate immune cell behavior and is thus likely to be a key player in immune dysregulation. NFkB is a family of TFs containing 5 subunits (cRel, RelA, p50, p52, and RelB) that combinatorically interact to form 15 possible dimeric species to regulate gene expression. Although, NFkB is well studied, there remains a paucity of research investigating the function of individual NFkB dimers in immune cell activation. Preliminary analysis supports the central hypothesis that each dimer of NFkB has a unique biological function. Understanding the role of individual NFkB dimers could open avenues for the precise modulation of pro-inflammatory gene expression. Further, my data supports the hypothesis that cRel homodimers are necessary for the selective regulation of a small group of genes including Il12b; and p50 homodimers associate with a co-regulatory protein to activate key pro-inflammatory genes including Il6, Il1b and Lcn2. This research employs a bone marrow derived macrophage model system in combination with the extraction of primary peritoneal macrophages stimulated ex vivo with E. coli or in vivo with a cecal ligation and puncture model of sepsis, to define dimer specific roles of NFkB in macrophage activation and the molecular mechanisms underlying dimer specific gene regulation. This proposal will i) explore the mechanistic role of cRel homodimers in macrophage activation; and ii) investigate p50 homodimer specific gene regulation in macrophage activation. This research proposal focuses on cRel and p50 homodimers due to their potent activation downstream of viral and bacterial PRRs and their highly specific roles in immune cell activation. Upon completion, this research will provide novel insights into the regulatory logic employed by NFkB by revealing the underlying mechanisms that make dimer specific functions possible. Further, it could uncover specific therapeutic targets for immune system modulation to help slow or halt exaggerated immune cell activation seen in sepsis and ARDS.