AREG contributes to the diminished PICD of neonatal monocytes by EGFR-mediated apoptosis suppression

Platen, Christopher; Orlikowsky, Thorsten (Thesis advisor); Usadel, Björn (Thesis advisor); Bohrmann, Johannes (Thesis advisor)

Aachen (2018, 2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2018


Neonates are highly susceptible to microbial infections, resulting in nearly 1 million neonatal deaths per year. This predisposition is particularly referable to distinct differences between the immune system of adults and neonates. The process of inflammation is usually triggered as a protective response to infection or tissue injury. The accurate resolution of the inflammatory response is essential to return to tissue homeostasis since a prolonged inflammation with ongoing cytokine release can result in tissue damage, organ dysfunctions and mortality. This is of particular importance for newborns, since various neonatal diseases, including periventricular leukomalacia, necrotizing enterocolitis or bronchopulmonary dysplasia, are characterized by sustained inflammation. One factor known to control length and extent of the inflammatory response, is the regulation of effector cell apoptosis. A special type of apoptosis is the phagocytosis-induced cell death (PICD) of phagocytic immune cells. Previous studies demonstrated that PICD is less frequently triggered in neonatal monocytes than in adult monocytes. Hence, a rescue of monocyte PICD could be a potential therapeutic approach to target the sustained inflammation in neonates. The EGF receptor is capable of blocking the initiation of apoptosis upon activation by its specific ligands. One ligand is the cytokine amphiregulin (AREG), which is increasingly shedded in response to bacterial infection and was shown to mediate apoptosis resistance. Therefore, it was hypothesized that AREG might contribute to the reduced PICD of neonatal monocytes by affecting the apoptosis signaling of monocytes. In this study, a well-established in vitro E. coli infection model was used to analyze PICD in primary monocytes from peripheral (PBMO) or cord blood (CBMO). The results indicate that CBMO show remarkably higher AREG surface expression as well as soluble levels in response to infection which could function as anti-apoptotic stimulus. Flow cytometry experiments were conducted to analyze intrinsic and extrinsic apoptosis signaling in dependence on AREG. Strikingly, AREG was proven to affect both pathways through EGFR downstream trafficking. With regard to extrinsic apoptosis, AREG increases intracellular MMP-2 and MMP-9 levels through EGFR activation, whereby inducing cleavage of membrane-bound FasL. Intrinsic apoptosis signaling is supressed by AREG function via EGFR-mediated phosphorylation of ERK1/2, Akt and BAD, resulting in increased intracellular Bcl-2 and Bcl-XL levels and decreased cleavage of pro-caspase-9 and -3. Furthermore, the results indicate that AREG may have regulatory function on T cells, which are of special importance for the immune response as they perform key effector functions during inflammation. AREG potentially reduces T cell proliferation through the suppressed costimulatory capacity of monocytes, since surface expression of CD80, CD86 und HLA-DR was found downregulated. The temporary presence during the newborn period might identify AREG as endogenous immune regulator. Targeting inflammatory diseases in neonates is of central clinical relevance and a better understanding of the regulatory elements involved in PICD could be essential to find new therapeutic strategies. Inhibition of AREG rescued the PICD of neonatal monocytes, thereby identifying AREG as potential target to reduce sustained inflammation in neonates.