Contribution of Interferon-β to the Murine Macrophage Response to the Toll-like Receptor 4 Agonist, Lipopolysaccharide

Stefanie N Vogel,Raj Deonarain Newman,Karen E Thomas,Eleanor N Fish,Carole L Galligan

doi:10.1074/jbc.m604958200

Stefanie N Vogel, Raj Deonarain Newman + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m604958200

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Abstract

Interferon-beta (IFN-beta) has been identified as the signature cytokine induced via the Toll-like receptor (TLR) 4, "MyD88-independent" signaling pathway in macrophages stimulated by Gram-negative bacterial lipopolysaccharide (LPS). In this study, we analyzed the responses of macrophages derived from wild-type (IFN-beta(+/+)) mice or mice with a targeted mutation in IFN-beta (IFN-beta(-/-)) to the prototype TLR4 agonist, Escherichia coli LPS. A comparison of basal and LPS-induced gene expression (by reverse transcription-PCR, real-time PCR, and Affymetrix microarray analyses) resulted in the identification of four distinct patterns of gene expression affected by IFN-beta deficiency. Analysis of a subset of each group of differentially regulated genes by computer-assisted promoter analysis revealed putative IFN-responsive elements in all genes examined. LPS-induced activation of intracellular signaling molecules, STAT1 Tyr-701, STAT1 Ser-727, and Akt, but not p38, JNK, and ERK MAPK proteins, was significantly diminished in IFN-beta(-/-) versus IFN-beta(+/+) macrophages. "Priming" of IFN-beta(-/-) macrophages with exogenous recombinant IFN-beta significantly increased levels of LPS-induced gene expression for induction of monocyte chemotactic protein 5, inducible nitric-oxide synthase, IP-10, and IL-12 p40 mRNA, whereas no increase or relatively small increases were observed for IL-1beta, IL-6, monocyte chemotactic protein 1, and MyD88 mRNA. Finally, IFN-beta(-/-) mice challenged in vivo with LPS exhibited increased survival when compared with wild-type IFN-beta(+/+) controls, indicating that IFN-beta contributes to LPS-induced lethality; however, not to the extent that one observes in mice with more complete pathway deficiencies (e.g. TLR4(-/-) or TRIF(-/-) mice). Collectively, these findings reveal unanticipated regulatory roles for IFN-beta in response to LPS in vitro and in vivo.

Highlights

Not all Toll-like receptor (TLR) activate the pro-inflammatory response to the same extent
Escherichia coli LPS signaling through TLR4 resulted in a much broader pattern of gene expression in murine macrophages than was observed in cells stimulated with the TLR2 agonist, Porphyromonas gingivalis LPS
TLR2 appears to be limited to interaction with MyD88 and TIRAP, and fails to activate genes that are downstream of the MyD88-independent pathway

Summary

EXPERIMENTAL PROCEDURES

Reagents—Protein-free (Ͻ0.008%) E. coli K235 LPS was prepared by two cycles of hot phenol-water extraction as described previously [13]. Cells were treated with 10 ng/ml LPS for 1 or 3 h harvested using 2.4 units/ml Dispase (Sigma), and total RNA isolation was performed using the Qiagen RNeasy mini kit according to the manufacturer’s protocol for use in Affymetrix GeneChip௡ Microarray analyses (described below). MRNA Detection by RT-PCR and Southern Blot Analysis— Total RNA was isolated from macrophage cultures and reverse transcription-PCR (RT-PCR) was performed, as described previously [15]. The NCBI Gene identification numbers of select genes that were analyzed are as follows: Mx1 (17857), Ifit (15957), Ccl (Mcp5) (20293), Il6 (16193), Cxcl (IP-10) (15945), Rpgrip (77945), Ogn (18295), Gzme (14942), and Trim (94094) These genes were selected as being representative of the four groups of genes differentially regulated in wild-type and IFN␤Ϫ/Ϫ macrophages.

RESULTS

13 Ϯ 9a 631 Ϯ 146a

DISCUSSION