Distinct NF-κB and MAPK Activation Thresholds Uncouple Steady-State Microbe Sensing from Anti-pathogen Inflammatory Responses

Abstract

The innate immune system distinguishes low-level homeostatic microbial stimuli from those of invasive pathogens, yet we lack understanding of how qualitatively similar microbial products yield context-specific macrophage functional responses. Using quantitative approaches, we found that NF-κB and MAPK signaling was activated at different concentrations of a stimulatory TLR4 ligand in both mouse and human macrophages. Above a threshold of ligand, MAPK were activated in a switch-like manner, facilitating production of inflammatory mediators. At ligand concentrations below this threshold, NF-κB signaling occurred, promoting expression of a restricted set of genes and macrophage priming. Among TLR-induced genes, we observed an inverse correlation between MAPK dependence and ligand sensitivity, highlighting the role of this signaling dichotomy in partitioning innate responses downstream of a single receptor. Our study reveals an evolutionarily conserved innate immune response system in which danger discrimination is enforced by distinct thresholds for NF-κB and MAPK activation, which provide sequential barriers to inflammatory mediator production.

Figure 1. TLR4 stimulation yields a switch-like inflammatory threshold

(A) TLR ligation induces inflammatory cytokine production via activation of NF-kB and MAPK (p38, Erk, and Jnk). (B) BMDM, derived from C57BL/6 unless otherwise noted, were stimulated with the indicated concentration of Kdo2-LipidA (KLA) and analyzed by flow cytometry or qPCR. (C–E) BMDM were stained intracellularly for TNF and CCL3 after 4 hours in the presence of Brefeldin A (BFA). (D) TNF response profiles for multiple KLA doses, with the TNF MFI for cells within the positive gate shown on the right. (E) The normalized frequency of TNF+CCL3+ cells (%) or cytokine MFI for the entire population. Timecourse data for Tnf mRNA and TNF protein are shown in (F) and (G) respectively and these dose responses are compared by plotting the area under the curve (Area), plotted with the 4 hour cumulative TNF protein MFI from two independent experiments (H). Dose responses for the indicated inflammatory mediators are were determined using mRNA Area (I) or protein from 4-hour stimulation supernatants (J). Data are representative of 3 or more independent experiments. See also Figure S1.

Figure 2. Sub-threshold TLR stimulation primes macrophages without increasing ligand sensitivity

(A,B) BMDM were stimulated with the indicated concentration of KLA for 4 hours in the presence of BFA, with or without pre-stimulation with 0.03nM KLA, for 2h hours unless otherwise indicated (light blue histograms and lines). (C) Data from (B) were re-plotted as a percent of the maximum MFI measured across doses, for BMDM with or without pre-stimulation. Data are pooled from two independent experiments and representative of 3 experiments.

Figure 3. Restricted NF-κB mediated gene expression occurs below inflammatory threshold

(A–D) BMDM were stimulated with KLA and gene expression was assessed using RNAseq. (A) Venn diagram shows number of genes significantly upregulated at 1 or 2 hours, relative to 0nM KLA stimulated cells (log2 fold change > 0.5 and 1% FDR). (B) DiRE transcription factor motif analysis for the indicated sets of genes, upregulated by both KLA concentrations or exclusively by 10nM (log2 fold change > 0.5 and 1% FDR); importance is a product of motif occurrence (fraction of regulatory elements containing motif) and weight score (motif prevalence compared to background gene list). (C) Relative expression for upregulated genes, significantly changed by both 0.03nM and 10nM KLA (log2 fold change > 0.5 and 1% FDR). RNAseq was performed on pooled RNA from 3 biological replicates, for 2 independent experiments. See also Figure S2.

Figure 4. Distinct NF-κB and MAPK signaling thresholds downstream of a common input

BMDM were stimulated with the indicated concentration of KLA and signaling intermediates were analyzed by flow cytometry. (A) Representative flow cytometry data (shaded histograms represent 0nM KLA stimulated cells) and schematic of data analysis. (B, D, F) MFI are plotted for each concentration of KLA over time and (C, E, G) the peak/maximum MFI (Peak) and area under the curve (Area) for each concentration of ligand was extracted from time course data and plotted over the dose response for TNF. Data in (A, B, D, F) are from one of 4 independent experiments pooled in (C, E, G), showing mean and standard deviation. See also Figure S3 and Figure S4.

Figure 5. Sub-threshold gene induction inversely correlates with MAPK dependence

BMDM were stimulated with KLA and mRNA expression for 40 TLR-induced genes was measured using high-throughput qPCR. (A) Example timecourse data showing fold change in response to the indicated concentration of KLA, with area under the curve for 0.1nM shaded in blue. (B) Ligand sensitivities were determined using the area under the curve from timecourse experiments; plots show the distribution of forty genes (left plot) or responses for example genes with high or low relative expression at 0.1nM KLA (middle or right plot, respectively). (C) MAPK dependence was calculated based on the percentage of induction lost, comparing the 10nM fold change at peak timepoint (1h or 2h), with and without MAPK inhibition (MAPKi), MEK1/2 inhibitor U0126 and p38 inhibitor SB203580. (D) The MAPK dependence for the 40 genes assessed is shown as compared to relative induction at 0.1nM for each gene. Statistical significance for the slope of the regression line was determined using an F test and groups were compared using an unpaired t test, for the left and right plot respectively, with a statistical significance cutoff of p≤0.05. Data are representative of two independent experiments. See also Figure S5.

Figure 6. Dysregulated Erk activity is coincident with aberrant TNF production in RAW macrophages

BMDM or RAW were stimulated with the indicated concentration of KLA and signaling intermediates or TNF expression were analyzed by flow cytometry. (A) Erk phosphorylation or IκBα degradation plotted as a percent of maximum response for each cell type. (B) The peak/maximum MFI for each concentration of ligand was extracted from IκBα degradation and Erk phosphorylation time course data, normalized, and plotted over the dose response for TNF production. Select BMDM data are re-plotted from Figure 4, with curves representing the mean of 4 or 3 independent experiments for BMDM or RAW, respectively. (C–E) Duplicate wells of BMDM or RAW were stimulated for 4 hours in the presence of BFA, with or without MEK1/2 inhibitor U0126 or p38 inhibitor SB203580. All in vitro data are representative of 3 or more independent experiments. (F,G) C57Bl/6 mice were injected i.p. with the indicated dose of KLA, data are pooled from two independent experiments (n=7). Serum TNF (F) was assessed at the indicated times and frequency of Ly6G+ neutrophils in peritoneal lavage was assessed at 2 hours (G). (H) Balb/c mice were injected i.p. with 2.5×10⁶ CFSE labeled RAW cells just prior to administration of KLA, as above, and peritoneal macrophages and serum TNF were quantified 1h later; serum TNF levels pooled from 3 independent experiments (n=7, 4, 6, 9 and 6, from left to right). Box and whiskers represent 25^th to 75^th percentiles, and minimum to maximum values, respectively, and stars represent statistical significance based on ordinary one-way ANOVA, versus control (* p≤0.05; ** p≤0.01; *** p≤0.001, **** p≤0.0001). See also Figure S6.

Figure 7. Distinct TLR-induced NF-κB and MAPK signaling thresholds and tight dose-dependent TNF regulation in macrophages from diverse human subjects

(A–B) Monocyte-derived macrophages from 6 independent healthy donors were stimulated with TLR4 ligand KLA for 4 hours in the presence of BFA. The frequency of TNF positive cells or % maximum MFI for TNF expression is plotted for each individual. (C) Macrophages from two healthy donors were stimulated with varying concentrations of KLA and signaling intermediates then analyzed by flow cytometry. The area under the curve for each concentration of ligand was extracted from time course data of IκBα degradation, p38 phosphorylation, or Erk phosphorylation and plotted over the dose response for TNF production. TNF dose responses are representative of 15 individual donors tested in 3 independent experiments. Signaling responses are representative of 4 individual donors tested in 2 independent experiments. See also Figure S7.