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 IFNγ inducing activity in the conditioned media from LPS-stimulated monocytes, which was not due to the prototypical IFNγ inducing cytokine, Interleukin-18 (IL-18). Via neutralization, immunodepletion and size-fractionation experiments, we uncovered that the IFNγ inducing activity was due to the synergistic action of the monocyte-derived cytokines Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNFα). Moreover, our data suggested that the prototypical IFNγ 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 IFNγ production in the monocyte conditioned media. However, when the target IFNγ producing KG-1 cell line was directly stimulated with the recombinant forms of IL-18, IL-1β and TNFα, we found that IL-1β and IL-18 could both synergize with TNFα for IFNγ production. Since IL-1β 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-1β and IL-18 receptors (IL-1R and IL-18R, respectively) are crucial for the observed synergy between IL-1 cytokines and TNFα.
In the second part of this dissertation, we tested the role of the transcriptional regulator, IκBζ, in IFNγ production in response to IL-1 and TNFα combined stimuli. IκBζ 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 IκBζ expression, we uncovered that IκBζ has a crucial role in IL-1/TNFα-induced IFNγ production. IκBζ is known to regulate transcription by acting as a co-factor for the transcription factor, NFκB. Our findings indicated that IL-1/TNFα combined stimuli induces strong NFκB binding to two of three putative NFκB binding sites present in the IFNγ promoter and first intron. This data suggested that IFNγ production in response to IL-1/TNFα combined stimuli may be due to IκBζ/NFκB-mediated transcription of the IFNγ gene.
In summary, our studies revealed a novel synergistic role for IL-1 cytokines and TNFα in IFNγ production that is mediated by the transcriptional regulator, IκBζ. Moreover, given the primary response nature of IL-1 cytokines and TNFα, our findings may have important implications in understanding the mechanisms leading to IFNγ production during the initial stages of innate immune responses.