A novel role for IL-1 cytokines and TNFalpha in IFNgamma production, which is mediated by IkappaBzeta

The human immune response to pathogen infection involves a complex network of immune cells, sensor molecules and signaling molecules that fight the invading organism and can lead to its elimination. Lipopolysaccharide (LPS) is an important component of the outer membrane of Gram-negative bacteria that elicits potent host immune responses leading to inflammation. Monocytes and macrophages are some of the main responders to LPS via a surface expressed sensor molecule known as Toll-like receptor 4 (TLR4). LPS recognition via TLR4 leads to signaling events that allow the cell to re-arrange its gene expression profile and produce inflammatory molecules, such as pro-inflammatory cytokines, which have a wide range of immune functions.;The first part of this dissertation describes the early inflammatory response, in terms of cytokine release, of human primary monocytes to LPS. Specifically, we detected an IFNgamma inducing activity in the conditioned media from LPS-stimulated monocytes, which was not due to the prototypical IFNgamma inducing cytokine, Interleukin-18 (IL-18). Via neutralization, immunodepletion and size-fractionation experiments, we uncovered that the IFNgamma inducing activity was due to the synergistic action of the monocyte-derived cytokines Interleukin-1beta (IL-1beta) and Tumor Necrosis Factor-alpha (TNFalpha). Moreover, our data suggested that the prototypical IFNgamma inducing cytokine, IL-18, might have been bound to an inhibitory protein, released in sub-optimal amounts, or both. Hence, the lack of IL-18-mediated IFNgamma production in the monocyte conditioned media. However, when the target IFNgamma producing KG-1 cell line was directly stimulated with the recombinant forms of IL-18, IL-1beta and TNFalpha, we found that IL-1beta and IL-18 could both synergize with TNFalpha for IFNgamma production. Since IL-1beta and IL-18 belong to the same family of cytokines (the IL-1 family) and share similar signaling pathways, we hypothesized that common signaling events downstream of the IL-1beta and IL-18 receptors (IL-1R and IL-18R, respectively) are crucial for the observed synergy between IL-1 cytokines and TNFalpha.;In the second part of this dissertation, we tested the role of the transcriptional regulator, IkappaBzeta, in IFNgamma production in response to IL-1 and TNFalpha combined stimuli. IkappaBzeta is known to be induced downstream of the IL-1R. We showed for the first time that it can also be induced downstream of the IL-18R. Importantly, by silencing IkappaBzeta expression, we uncovered that IkappaBzeta has a crucial role in IL-1/TNFalpha-induced IFNgamma production. IkappaBzeta is known to regulate transcription by acting as a co-factor for the transcription factor, NFkappaB. Our findings indicated that IL-1/TNFalpha combined stimuli induces strong NFkappaB binding to two of three putative NFkappaB binding sites present in the IFNgamma promoter and first intron. This data suggested that IFNgamma production in response to IL-1/TNFalpha combined stimuli may be due to IkappaBzeta/NFkappaB-mediated transcription of the IFNgamma gene.;In summary, our studies revealed a novel synergistic role for IL-1 cytokines and TNFalpha in IFNgamma production that is mediated by the transcriptional regulator, IkappaBzeta. Moreover, given the primary response nature of IL-1 cytokines and TNFalpha, our findings may have important implications in understanding the mechanisms leading to IFNgamma production during the initial stages of innate immune responses.