Interrogating the role of transmembrane TNF (tmTNF) in TLR2 or TLR4 activated macrophages during Staphylococcus aureus infection

Abstract

Tumor Necrosis Factor alpha (TNF) is a cytokine involved in diverse cellular processes such as cell survival, proliferation, differentiation, immune defense, and cell death. TNF produced during an immune response is typically associated with the generation and propagation of inflammation. This association with inflammation is particularly strong for soluble TNF (sTNF), one of two bioactive TNF protein forms, but not transmembrane TNF (tmTNF), the second bioactive TNF protein form. Typically, tmTNF is more closely associated with anti- inflammatory or proliferative responses. As a result, much less is known about the role and extent to which tmTNF participates in host immune responses, particularly as it pertains to host defense. During infection, macrophages are known to secrete large amounts of TNF in response to pathogen detection via Toll-Like-Receptor (TLR) stimulation. In this dissertation research, we investigated the role of sTNF and tmTNF on macrophage priming following TLR2 or TLR4 stimulation and how this priming influenced macrophage ability to clear a subsequent Staphylococcus aureus infection. In vitro cell culture assays demonstrated that TLR stimulated WT bone marrow derived macrophages (BMDMs) became significantly more efficient at clearing a subsequent S. aureus infection. Transcriptomic and protein analysis revealed upregulation of both sTNF and tmTNF following TLR stimulation. We demonstrated that depletion of sTNF, but not tmTNF, during TLR stimulation did not influence BMDM priming which led to improved clearance of a subsequent bacterial infection. Conversely, supplementing exogenous sTNF to TLR2 stimulated TNF-/- BMDMs prior to infection did not improve S. aureus clearance. This indicated that sTNF signaling was not required for BMDM priming. Further, this suggested that TLR-induced tmTNF priming could contribute to improved S. aureus clearance. TmTNF preferentially signals through TNFR2. Indeed, blocking TNFR2 following TLR2 stimulation led to increased overall S. aureus bacterial burden. To mechanistically elucidate how tmTNF could be priming BMDMs prior to S. aureus infection, we investigated the potential of tmTNF reverse signaling. These studies provide valuable insight into the differential involvement of sTNF and tmTNF signaling following TLR stimulation of BMDMs which has implications for downstream response to infection.