Identification of 34 novel proinflammatory proteins in a genome-wide macrophage functional screen

Abstract

Signal transduction pathways activated by Toll-like Receptors and the IL-1 family of cytokines are fundamental to mounting an innate immune response and thus to clearing pathogens and promoting wound healing. Whilst mechanistic understanding of the regulation of innate signalling pathways has advanced considerably in recent years, there are still a number of critical controllers to be discovered. In order to characterise novel regulators of macrophage inflammation, we have carried out an extensive, cDNA-based forward genetic screen and identified 34 novel activators, based on their ability to induce the expression of cxcl2. Many are physiologically expressed in macrophages, although the majority of genes uncovered in our screen have not previously been linked to innate immunity. We show that expression of particular activators has profound but distinct impacts on LPS-induced inflammatory gene expression, including switch-type, amplifier and sensitiser behaviours. Furthermore, the novel genes identified here interact with the canonical inflammatory signalling network via specific mechanisms, as demonstrated by the use of dominant negative forms of IL1/TLR signalling mediators.

Figure 1. Flow chart of the functional screen.

A) shows the workflow of the screen. B) shows an example of the analysis performed on a plate comprising 96 pools of 12. The hypothesis that individuals pools differ from the plate mean is tested using a linear model. Three pools (P1, P2, P3) give signal over the −logp>4 cut off used. Breakdown of pool P2 is shown in C) one constituent clone (#8) gives high-level induction. Dots represent results of four independent replicates, performed for each clone.

Figure 2. Activation of proinflammatory promoters by library hits and their impact on LPS induced cxcl2 activation in Raw 264.7 cells.

Induction of three proinflammatory promoters (ifnb, cxcl2 and Lcn2) by plasmids recovered from the library screen and positive controls are shown. Results are derived from 2 (for ifnb and lcn2) and between 3 and 13 independent experiments (for cxcl2), all performed in triplicate in each experiment. A) Results are expressed as log fold increase of normalised promoter activity over background and 95% confidence intervals. B) and C) show bivariate plots illustrating activity of the various molecules on the cxcl2, lcn2 and ifnb reporters. Effect estimates for molecules derived from the screen are marked as circles, and known components of signalling system as triangles. D–F) Raw 264.7 cells were transfected with the cxcl2-pLuc and EF1-rLuc reporters, as described in the methods, as well as with 60 ng/well expression plasmid, encoding known (TRAF6) (D) or novel proinflammatory molecules (E and F). The impact of over-expressed proinflammatory mediators on LPS induced cxcl2 expression was tested. The activity profile of the cDNAs tested were classified according to three distinct patterns, as exemplified in D–F and shown in G). Genes highlighted in bold encode known components of inflammatory signal transduction (controls).

Figure 3. Screen hits interact with the canonical proinflammatory signalling network via distinct molecular mechanisms.

A) Raw 264.7 cells were transfected with the cxcl2-pLuc and EF1-rLuc reporters, as described in the methods, as well as with 30 ng/well expression plasmid, encoding for dominant negative (DN) mutants of known proinflammatory molecules (MyD88, TRAM, TIRAP, TRIF, IRAK1, TRAF6 and Ras, respectively). LPS (100 ng/ml) was used as a positive control (6 hrs stimulation) to test for the inhibitory activity of the DN constructs used. Data are derived from three independent experiments, as described in Methods. *p<0.05 , **p<0.01, ***p<0.001 B) summarises the interactions observed in A.

Figure 4. Selected screen hits are required for TLR4 but not for TLR7/8 induced cxcl2 induction.

Raw 264.7 cells were transfected with the cxcl2-pLuc and EF1-rLuc reporters, as described in the methods, as well as with 10 pmol siRNA (ON-TARGET plus SMART pool, Dharmacon) aginst selected hits that are endogenously expressed in these cells. The impact of siRNA knockdown on A) LPS (20 ng/ml) or B) CL075 (2 µg/ml) induced cxcl2 reporter activation was measured after 6 hrs stimulation. White bars: vehicle, grey bars: agonist added. Data was analysed by two-way ANOVA modelling effect of agonist (LPS or CL075) and siRNA (four categories). Significance was determined from contrast estimates from the model (***p<0.001, ****p<0.0001). A representative experiment of three, with similar results, is shown.