Network dynamics determine the autocrine and paracrine signaling functions of TNF.

A hallmark of the inflammatory response to pathogen exposure is the production of tumor necrosis factor (TNF) that coordinates innate and adaptive immune responses by functioning in an autocrine or paracrine manner. Numerous molecular mechanisms contributing to TNF production have been identified, but how they function together in macrophages remains unclear. Here, we pursued an iterative systems biology approach to develop a quantitative understanding of the regulatory modules that control TNF mRNA synthesis and processing, mRNA half-life and translation, and protein processing and secretion. By linking the resulting model of TNF production to models of the TLR-, the TNFR-, and the NFκB signaling modules, we were able to study TNF's functions during the inflammatory response to diverse TLR agonists. Contrary to expectation, we predicted and then experimentally confirmed that in response to lipopolysaccaride, TNF does not have an autocrine function in amplifying the NFκB response, although it plays a potent paracrine role in neighboring cells. However, in response to CpG DNA, autocrine TNF extends the duration of NFκB activity and shapes CpG-induced gene expression programs. Our systems biology approach revealed that network dynamics of MyD88 and TRIF signaling and of cytokine production and response govern the stimulus-specific autocrine and paracrine functions of TNF.

B-cell survival and development controlled by the coordination of NF-κB family members RelB and cRel.

Targeted deletion of BAFF causes severe deficiency of splenic B cells. BAFF-R is commonly thought to signal to nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase dependent noncanonical NF-κB RelB. However, RelB-deficient mice have normal B-cell numbers. Recent studies showed that BAFF also signals to the canonical NF-κB pathway, and we found that both RelB and cRel are persistently activated, suggesting BAFF signaling coordinates both pathways to ensure robust B-cell development. Indeed, we report now that combined loss of these 2 NF-κB family members leads to impaired BAFF-mediated survival and development in vitro. Although single deletion of RelB and cRel was dispensable for normal B-cell development, double knockout mice displayed an early B-cell developmental blockade and decreased mature B cells. Despite disorganized splenic architecture in Relb(-/-)cRel(-/-) mice, generation of mixed-mouse chimeras established the developmental phenotype to be B-cell intrinsic. Together, our results indicate that BAFF signals coordinate both RelB and cRel activities to ensure survival during peripheral B-cell maturation.

A multi-scale approach reveals that NF-κB cRel enforces a B-cell decision to divide.

Understanding the functions of multi-cellular organs in terms of the molecular networks within each cell is an important step in the quest to predict phenotype from genotype. B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically. Based on tracking single-cell time-lapse trajectories of hundreds of B cells, single-cell transcriptome, and immunofluorescence analyses, we constructed an agent-based multi-modular computational model to simulate lymphocyte population dynamics in terms of the molecular networks that control NF-κB signaling, the cell cycle, and apoptosis. Combining modeling and experimentation, we found that NF-κB cRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells. But as cRel deficiency causes growing B cells to die at similar rates to non-growing cells, our analysis reveals that the phenomenological decision model of wild-type cells is rooted in a biased race of cell fates. We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks.

Analysis of the RelA:CBP/p300 interaction reveals its involvement in NF-κB-driven transcription.

NF-κB plays a vital role in cellular immune and inflammatory response, survival, and proliferation by regulating the transcription of various genes involved in these processes. To activate transcription, RelA (a prominent NF-κB family member) interacts with transcriptional co-activators like CREB-binding protein (CBP) and its paralog p300 in addition to its cognate κB sites on the promoter/enhancer regions of DNA. The RelA:CBP/p300 complex is comprised of two components--first, DNA binding domain of RelA interacts with the KIX domain of CBP/p300, and second, the transcriptional activation domain (TAD) of RelA binds to the TAZ1 domain of CBP/p300. A phosphorylation event of a well-conserved RelA(Ser276) is prerequisite for the former interaction to occur and is considered a decisive factor for the overall RelA:CBP/p300 interaction. The role of the latter interaction in the transcription of RelA-activated genes remains unclear. Here we provide the solution structure of the latter component of the RelA:CBP complex by NMR spectroscopy. The structure reveals the folding of RelA-TA2 (a section of TAD) upon binding to TAZ1 through its well-conserved hydrophobic sites in a series of grooves on the TAZ1 surface. The structural analysis coupled with the mechanistic studies by mutational and isothermal calorimetric analyses allowed the design of RelA-mutants that selectively abrogated the two distinct components of the RelA:CBP/p300 interaction. Detailed studies of these RelA mutants using cell-based techniques, mathematical modeling, and genome-wide gene expression analysis showed that a major set of the RelA-activated genes, larger than previously believed, is affected by this interaction. We further show how the RelA:CBP/p300 interaction controls the nuclear response of NF-κB through the negative feedback loop of NF-κB pathway. Additionally, chromatin analyses of RelA target gene promoters showed constitutive recruitment of CBP/p300, thus indicating a possible role of CBP/p300 in recruitment of RelA to its target promoter sites.

IκBε is a key regulator of B cell expansion by providing negative feedback on cRel and RelA in a stimulus-specific manner.

The transcription factor NF-κB is a regulator of inflammatory and adaptive immune responses, yet only IκBα was shown to limit NF-κB activation and inflammatory responses. We investigated another negative feedback regulator, IκBε, in the regulation of B cell proliferation and survival. Loss of IκBε resulted in increased B cell proliferation and survival in response to both antigenic and innate stimulation. NF-κB activity was elevated during late-phase activation, but the dimer composition was stimulus specific. In response to IgM, cRel dimers were elevated in IκBε-deficient cells, yet in response to LPS, RelA dimers also were elevated. The corresponding dimer-specific sequences were found in the promoters of hyperactivated genes. Using a mathematical model of the NF-κB-signaling system in B cells, we demonstrated that kinetic considerations of IκB kinase-signaling input and IκBε's interactions with RelA- and cRel-specific dimers could account for this stimulus specificity. cRel is known to be the key regulator of B cell expansion. We found that the RelA-specific phenotype in LPS-stimulated cells was physiologically relevant: unbiased transcriptome profiling revealed that the inflammatory cytokine IL-6 was hyperactivated in IκBε(-/-) B cells. When IL-6R was blocked, LPS-responsive IκBε(-/-) B cell proliferation was reduced to near wild-type levels. Our results provide novel evidence for a critical role for immune-response functions of IκBε in B cells; it regulates proliferative capacity via at least two mechanisms involving cRel- and RelA-containing NF-κB dimers. This study illustrates the importance of kinetic considerations in understanding the functional specificity of negative-feedback regulators.

Limited specificity of IRF3 and ISGF3 in the transcriptional innate-immune response to double-stranded RNA.

The innate immune response is largely initiated by pathogen-responsive activation of the transcription factor IRF3. Among other target genes, IRF3 controls the expression of IFN-ß, which triggers the activation of the transcription factor ISGF3 via the IFNAR. IRF3 and ISGF3 have been reported to control many of the same target genes and together, control the antimicrobial innate-immune program; however, their respective contributions and specificities remain unclear. Here, we used genomic technologies to characterize their specificity in terms of their physical DNA-binding and genetic function. With the use of ChiP-seq and transcriptomic measurements in WT versus ifnar(-/-) versus ifnar(-/-)irf3(-/-) macrophages responding to intracellular dsRNA, we confirmed the known ISGF3 DNA-binding motif and further specified a distinct IRF3 consensus sequence. The functional specificity of IRF3 is particularly pronounced in cytokine/chemokine regulation; yet, even in the control of IFN-ß, that specificity is not absolute. By mathematically modeling IFN-ß production within an abstracted tissue layer, we find that IRF3 versus ISGF3 specificity may be critical to limiting IFN-ß production and ISGF3 activation, temporally and spatially, but that partial overlap in their specificity is tolerable and may enhance the effectiveness of the innate-immune response.

A pathway switch directs BAFF signaling to distinct NFκB transcription factors in maturing and proliferating B cells.

BAFF, an activator of the noncanonical NFκB pathway, provides critical survival signals during B cell maturation and contributes to B cell proliferation. We found that the NFκB family member RelB is required ex vivo for B cell maturation, but cRel is required for proliferation. Combined molecular network modeling and experimentation revealed Nfkb2 p100 as a pathway switch; at moderate p100 synthesis rates in maturing B cells, BAFF fully utilizes p100 to generate the RelB:p52 dimer, whereas at high synthesis rates, p100 assembles into multimeric IκBsome complexes, which BAFF neutralizes in order to potentiate cRel activity and B cell expansion. Indeed, moderation of p100 expression or disruption of IκBsome assembly circumvented the BAFF requirement for full B cell expansion. Our studies emphasize the importance of p100 in determining distinct NFκB network states during B cell biology, which causes BAFF to have context-dependent functional consequences.