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An evolutionarily conserved innate immunity protein interaction network - PubMed

  • ️Tue Jan 01 2013

An evolutionarily conserved innate immunity protein interaction network

Lesly De Arras et al. J Biol Chem. 2013.

Abstract

The innate immune response plays a critical role in fighting infection; however, innate immunity also can affect the pathogenesis of a variety of diseases, including sepsis, asthma, cancer, and atherosclerosis. To identify novel regulators of innate immunity, we performed comparative genomics RNA interference screens in the nematode Caenorhabditis elegans and mouse macrophages. These screens have uncovered many candidate regulators of the response to lipopolysaccharide (LPS), several of which interact physically in multiple species to form an innate immunity protein interaction network. This protein interaction network contains several proteins in the canonical LPS-responsive TLR4 pathway as well as many novel interacting proteins. Using RNAi and overexpression studies, we show that almost every gene in this network can modulate the innate immune response in mouse cell lines. We validate the importance of this network in innate immunity regulation in vivo using available mutants in C. elegans and mice.

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Figures

FIGURE 1.
FIGURE 1.

An innate immunity protein interaction network. The protein interaction map depicts human proteins with known homologous protein-protein interactions in C. elegans, humans, or Drosophila (list of protein-protein interactions with references is given in Table 1 and a list of gene homologs is provided in

supplemental Table 2

). Each line between proteins indicates a single reported interaction; multiple lines indicate multiple reports of that interaction. Solid black bar indicates a well established biochemical interaction with multiple reports. Proteins with an RNAi-induced phenotype in C. elegans (decreased antimicrobial production (12)) or mouse macrophages (decreased LPS-induced IL-6 production, see Fig. 2 and Ref. 12) are color-coded as indicated. Proteins in the canonical TLR4 LPS response pathway that are already known to regulate innate immunity are labeled with red boxes around the protein name.

FIGURE 2.
FIGURE 2.

Most genes in the innate immunity protein interaction network affect LPS-induced IL-6 production in the J774A.1 mouse macrophage cell line. A, pools of four siRNA duplexes per gene were transfected into the mouse macrophage cell line J774A.1; cells were stimulated with 20 ng/ml LPS for 6 h, and IL-6 production was monitored by ELISA on cell supernatants. IL-6 production was normalized relative to a control pool of siRNA duplexes (CT1, Dharmacon nontargeting siRNA pool). CT2 is a second negative control (Dharmacon nontargeting siRNA 1). TLR4, the LPS receptor, is presented as a positive control. Two genes in this network (Macf1 and Siah1a) were inhibited previously; the data for these two genes from this prior publication (12) is presented at the end of the panel. B depicts the effects on viability of the indicated siRNA treatments normalized so that viability of control siRNA was equal to 1. C, depicts the results of qPCR, which was used to monitor RNA knockdown of the indicated genes. Asterisks indicate siRNA treatments that induced IL-6 levels (A) or gene knockdown (C) that were significantly different from the controls (p < 0.05). No viability measurements (B) were statistically significantly different from control.

FIGURE 3.
FIGURE 3.

SF3A1 and GOLGA4 regulate LPS-induced IL-6 release in the J774A.1 mouse macrophage cell line. To confirm the results generated by the pools of siRNA duplexes in Fig. 2 for the two genes that induced the strongest phenotypes, each of four individual siRNA duplexes (labeled A–D) were used to inhibit either Sf3a1 or Golga4. The figure depicts LPS-induced IL-6 production following siRNA treatment (20 ng/ml LPS for 6 h). IL-6 is off scale as indicated by the arrow above Golga4 siRNA (D) (733 ± 57%). Asterisks indicate siRNA treatments that induced IL-6 levels that were statistically different from the control (p < 0.05).

FIGURE 4.
FIGURE 4.

Confirmation that SF3A1 and GOLGA4 regulate LPS-induced IL-6 production using a second mouse macrophage cell line. A and B, indicated pools of siRNA duplexes were transfected into the RAW264.7 mouse macrophage cell line; cells were simulated with LPS (20 ng/ml for 6 h), and IL-6 production in cell supernatants was monitored by ELISA. C depicts the results of qPCR, which was used to monitor RNA knockdown of the indicated genes in B. D, indicated pools of siRNA duplexes were transfected into the RAW264.7 mouse macrophage cell line; cells were stimulated with LPS (20 ng/ml for 6 h), and IL-6 RNA production was monitored by qPCR. Asterisks indicate siRNA treatments that induced IL-6 protein levels (B), gene knockdown (C), or IL-6 RNA levels (D) that were statistically different from the controls (p < 0.05).

FIGURE 5.
FIGURE 5.

Mutation of innate immunity network genes in C. elegans alters nematode host defense. Depicted are survival curves for the indicated mutant strains exposed to the pathogen P. aeruginosa PA14 (left panels), nonpathogenic E. coli OP50 (middle panels), or heat-killed E. coli OP50. N2 (wild type nematodes) is depicted in black; the indicated mutants are depicted in red. The tested alleles and further statistical data (medians, n, and p values) are listed in

supplemental Table 3

.

FIGURE 6.
FIGURE 6.

MACF1 inhibits PAMP-induced cytokine production in RAW264.7 cells and in vivo. A, pool of four siRNA duplexes targeting either Macf1 or a nontargeting control siRNA duplex pool were transfected into the mouse macrophage cell line RAW264.7; cells were stimulated with 20 ng/ml LPS for 6 h, and IL-6 production was monitored by ELISA. B, RAW264.7 cells were transfected with plasmids overexpressing either Macf1 or CAT, an NFκB-AP1-luciferase reporter, and the SV40-rluc control plasmid. After transfection, the cells were stimulated (or not) with LPS as indicated. Firefly luciferase activity was monitored and normalized relative to the Renilla luciferase control. C and D, BMDMs from Macf1flox/flox;Lyzs-cre mice and control BMDM from Macf1+/+;Lyzs-cre mice were exposed to either 2 ng/ml LPS or 20 ng/ml PAM3CSK4 for 6 h, and IL-6 production was monitored by ELISA. n = 9, p < 0.05 for both PAMPs. Asterisks indicate data that was significantly different from the control (p < 0.05).

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