Flipping the NF-κB Switch in Macrophages

Rewiring of the NF-κB regulatory circuit at a critical dose of LPS promotes a full innate immune response. Lower model and time-lapse images: Subcritical doses of LPS induce predominantly negative feedback genes and genes with conflicting functional consequences, preventing a coherent response in macrophages. Upper model and time-lapse images: Above a critical dose of LPS, Ikaros-dependent amplification of NF-κB becomes a dominant positive feedback loop, which overcomes the various negative feed

Rewiring of the NF-κB regulatory circuit at a critical dose of LPS promotes a full innate immune response. Lower model and time-lapse images: Subcritical doses of LPS induce predominantly negative feedback genes and genes with conflicting functional consequences, preventing a coherent response in macrophages. Upper model and time-lapse images: Above a critical dose of LPS, Ikaros-dependent amplification of NF-κB becomes a dominant positive feedback loop, which overcomes the various negative feedback loops and enables a full-blown innate immune response.

A critical component of the innate immune system, macrophages respond to diverse microbes by recognizing certain molecular patterns, such as the Gram-negative bacteria product lipopolysaccharide (LPS), via Toll-like receptors. Receptor activation stimulates a complex signaling network that involves, among others, the NF-κB pathway. The complexity of this network has hampered researchers’ understanding of how macrophages resolve conflicting signals to determine when to mount an immune response.

To begin teasing apart the intricacies of macrophage activity, Gordon Hager, Ph.D., Chief of CCR’s Laboratory of Receptor Biology and Gene Expression and Myong-Hee Sung, Ph.D., a Staff Scientist in his lab, working with Iain Fraser, Ph.D., of the National Institute of Allergy and Infectious Diseases, and their colleagues decided to examine LPS-induced NF-κB activity in individual macrophages. Unlike population-based approaches, studies of single cells are more likely to reveal a pathway’s true dynamics. No prior single-cell studies of NF-κB signaling in immune cells had been conducted.

The researchers devised a reporter system to simultaneously monitor NF-κB nuclear localization and transcription. They infected a macrophage cell line with a lentivirus that co-expressed GFP-tagged RelA, a subunit of NF-κB, under the control of the RelA promoter and a rapidly degraded mCherry fluorescent protein under the control of the TNF-α promoter, an NF-κB target. When treated with a high dose of LPS, the infected cells exhibited maximum nuclear localization of RelA after 1 hour that decreased within 5 hours. After this initial phase, cells showed varying levels of RelA nuclear localization. Expression of mCherry was also induced by a high dose of LPS and was sustained for several hours. The NF-κB kinetics the investigators observed were distinct from those of LPS-treated fibroblasts, suggesting previous models of NF-κB signaling may not apply to macrophages. They also found little correlation between nuclear NF-κB and mCherry production at any single point.

To understand how NF-κB dynamics reflect LPS signaling, the research team treated lentivirus-infected cells with various concentrations of LPS. In contrast to the response in nonimmune cells, which showed higher percentages of cells responding to greater doses of LPS, nearly all the macrophages responded regardless of the LPS dose. With increasing LPS, the scientists did find that the time to peak nuclear RelA decreased in the macrophages. However, the peak nuclear fluorescence intensity and the duration of the peak were the same at all but the lowest LPS dose.

Again, the investigators observed low correlation between the activity of the mCherry transcription reporter and RelA nuclear localization. When they looked closer at their data, the researchers found that the cells treated with the higher doses of LPS were responsible for this poor correlation. These cells maintained higher nuclear RelA even when the ratio of nuclear to total RelA returned to unstimulated levels. This change was due to an increase in total RelA, meaning that the nuclear translocation dynamics now became distinct from nuclear intensity. Using regression analysis, the scientists found that RelA nuclear occupancy, defined as the activity of nuclear NF-κB integrated over time, correlated best with mCherry reporter activity. Importantly, these two measures correlated at the single-cell level despite the variety of other transcription factors at work in the system, suggesting the central role of NF-κB in TNF-α expression in macrophages. These results also showed that increased RelA is linked to tight control of gene expression by NF-κB.

The researchers were surprised by the LPS-induced increase in GFP-RelA, since RelA levels were thought to be insensitive to stimuli, and tested whether LPS affected endogenous RelA. They found that, above a threshold dose, LPS increased RelA mRNA and protein in both the macrophage cell line and primary macrophages. Like other NF-κB family members that are direct targets of NF-κB, the investigators showed that RelA could also bind its own promoter, identifying a previously unknown NF-κB positive feedback loop.

Using microarrays, the scientists further explored macrophage responses above and below the threshold level of LPS that induced RelA. They identified two groups of genes: those induced by high and low LPS levels and those induced by only high LPS. The latter was enriched in select gene ontology categories that were consistent with an antibacterial response. In contrast, genes induced by both high and low LPS had opposing activities. These results suggest that the LPS dose threshold may correspond to the turning point at which macrophages commit to mounting an immune response.

The investigators then generated a mathematical model of LPS-induced NF-κB signaling that included the newly-identified positive feedback pathway. Their simulations showed that the positive feedback loop could overcome known negative feedback pathways regulating NF-κB with increasing doses of LPS. The switch from predominantly negative feedback to positive feedback allowed the cells to discriminate between various levels of LPS. To test their model experimentally, the researchers asked whether the transcription of LPS-induced, NF-κB-regulated genes was affected by a prior dose of LPS, a phenomenon known as hysteresis. Indeed, they found that macrophages treated with a high dose of LPS and switched to a low dose showed much higher mRNA levels for the specific genes than cells pretreated with a low dose. The presence of hysteresis supports the proposed model and the importance of positive feedback for sensing LPS levels.

Finally, the scientists tested whether another factor might be involved in RelA induction by LPS, given the dose dependence of the response. They performed a genome-wide siRNA screen to identify genes that affected LPS-induced GFP-RelA and mCherry production when knocked down. Combining data from the siRNA screen with the earlier microarray data, the researchers identified the transcription factor Ikaros. Ikaros bound the RelA promoter in response to high-dose LPS, and knocking out Ikaros prevented the positive feedback pathway, hampering LPS dose discrimination. The investigators examined macrophages from Ikaros knockout mice and found no LPS dose-dependent increase in RelA and reduced expression of innate immune genes usually induced by high-dose LPS. Because Ikaros is present even in untreated macrophages, basal and induced Ikaros may be important for the dose-dependent increase in RelA. Understanding this mechanism is an important topic for future research.

Together, these studies revealed a novel positive feedback pathway for switching on NF-κB in macrophages in response to physiologically relevant concentrations of LPS. This response is distinct from that of other cell types, such as fibroblasts, and depends on the transcription factor Ikaros. This multidisciplinary investigation exemplifies how a quantitative single-cell analysis, in combination with high throughput profiling approaches, can lead to novel findings, even from extensively studied molecular systems.

Summary Posted: 02/2014

Reference

Sung MH, Li N, Lao Q, Gottschalk RA, Hager GL, and Fraser IDC. Switching of the Relative Dominance Between Feedback Mechanisms in Lipopolysaccharide-Induced NF-κB Signaling. Science Signaling. January 14, 2014 PubMed Link