Cindy A. Salkowski

Signaling by Toll-Like Receptor 2 and 4 Agonists Results in Differential Gene Expression in Murine Macrophages

ABSTRACT Lipopolysaccharide (LPS) derived from the periodontal pathogenPorphyromonas gingivalis has been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation of P. gingivalis LPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by this P. gingivalis LPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages by Escherichia coli LPS. These data suggest that (i) P. gingivalis LPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.

Distinct Mutations in IRAK-4 Confer Hyporesponsiveness to Lipopolysaccharide and Interleukin-1 in a Patient with Recurrent Bacterial Infections

We identified previously a patient with recurrent bacterial infections who failed to respond to gram-negative LPS in vivo, and whose leukocytes were profoundly hyporesponsive to LPS and IL-1 in vitro. We now demonstrate that this patient also exhibits deficient responses in a skin blister model of aseptic inflammation. A lack of IL-18 responsiveness, coupled with diminished LPS and/or IL-1–induced nuclear factor–κB and activator protein-1 translocation, p38 phosphorylation, gene expression, and dysregulated IL-1R–associated kinase (IRAK)–1 activity in vitro support the hypothesis that the defect lies within the signaling pathway common to toll-like receptor 4, IL-1R, and IL-18R. This patient expresses a “compound heterozygous” genotype, with a point mutation (C877T in cDNA) and a two-nucleotide, AC deletion (620–621del in cDNA) encoded by distinct alleles of the IRAK-4 gene (GenBank/EMBL/DDBJ accession nos. AF445802 and AY186092). Both mutations encode proteins with an intact death domain, but a truncated kinase domain, thereby precluding expression of full-length IRAK-4 (i.e., a recessive phenotype). When overexpressed in HEK293T cells, neither truncated form augmented endogenous IRAK-1 kinase activity, and both inhibited endogenous IRAK-1 activity modestly. Thus, IRAK-4 is pivotal in the development of a normal inflammatory response initiated by bacterial or nonbacterial insults.

Induction of early inflammatory gene expression in a murine model of nonresuscitated, fixed-volume hemorrhage.

The etiology of many end-organ problems associated with hemorrhage has been attributed to the inflammatory response to hemorrhage. In a murine model of nonresuscitated, fixed-volume hemorrhage, we sought to elucidate the role that hemorrhagic insult alone plays in the generation of the early inflammatory cascade. Differences could be appreciated as early as 1 h post-hemorrhage, with consistent differences detected by 3 h in all of the major cytokine genes studied. Significant upregulation of IL-1&bgr;, IL-6, TNF-&agr;, and IL-10 mRNA expression was observed in both the liver and lung samples of mice subjected to fixed-volume hemorrhage when compared with sham-hemorrhaged mice. The cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) genes also were upregulated in the livers and lungs of hemorrhaged mice. Finally, expression of the genes that encode the Toll-like receptors (TLR)-2 and -4 was increased by hemorrhage. Taken collectively, these data demonstrate that the initial inflammatory cascade associated with hemorrhage occurs within hours after the initial hemorrhagic event, and can be associated with significant modulation of expression of key pro- and anti-inflammatory cytokine, enzyme, and TLR genes, suggesting that these may be possible new therapeutic targets.

Impaired IFN-γ Production in IFN Regulatory Factor-1 Knockout Mice During Endotoxemia Is Secondary to a Loss of Both IL-12 and IL-12 Receptor Expression

Mice with a targeted mutation in the gene that encodes the transcription factor IFN regulatory factor-1 (IRF-1) were used to assess the contribution of IRF-1 to IL-12-dependent and IL-12-independent pathways of IFN-γ production. In response to LPS, IRF-1−/− mice produced less IL-12 p40, IL-12 p35, and IFN-γ mRNA in the liver than IRF-1+/+ mice. While pulmonary IFN-γ mRNA levels were also mitigated in IRF-1−/− mice, pulmonary IL-12 p40 and IL-12 p35 mRNA were not dysregulated. Circulating IL-12 p70 and IFN-γ levels were profoundly attenuated in LPS-challenged IRF-1−/− mice. Further analysis revealed a major deficiency in hepatic IL-12Rβ1 and IL-12Rβ2 mRNA expression as well as pulmonary IL-12Rβ1 mRNA expression in LPS-challenged IRF-1−/− mice. In vitro, IFN-γ up-regulated IL-12Rβ1 mRNA in macrophages from IRF-1+/+, but not IRF-1−/−, mice. IFN-γ-induced IL-12Rβ2 mRNA expression was also diminished in macrophages from IRF-1−/− mice. In contrast to IRF-1+/+ mice, administration of exogenous IL-12 to IRF-1−/− mice resulted in reduced serum IFN-γ and hepatic and pulmonary IFN-γ mRNA, demonstrating that loss of IL-12R results in diminished IL-12 responsiveness. While LPS-challenged IRF-1−/− mice also had reduced IL-15 mRNA levels, serum IL-18 responses were intact. Finally, induction of IRF-1 mRNA by LPS in livers of IFN-γ knockout mice were markedly attenuated, suggesting a feedback amplification loop. These studies indicate that IRF-1 deficiency disrupts both IL-12-dependent and -independent pathways of IFN-γ production and that IRF-1 is a critical transcription factor involved in the regulation of not only IL-12, but also IL-12R.

Effect of inflammatory and antiinflammatory stimuli on acyloxyacyl hydrolase gene expression and enzymatic activity in murine macrophages

Acyloxyacyl hydrolase (AOAH) is an enzyme found in macrophages and neutrophils that specifically cleaves the acyloxyacyl moieties of lipopolysaccharide (LPS), thus rendering it non-toxic for human cells. In the present study, we demonstrate that LPS augments AOAH mRNA expression (10-20-fold) in murine macrophages. Following LPS treatment (100 ng/m]), AOAH mRNA was induced by 2 h, peaked at 6 h, and was sustained over 72 h. Optimal induction of AOAH mRNA was observed with as little as 0.1 ng/ml LPS. LPS also induced a concomitant increase in AOAH enzymatic activity in cytosolic extracts from murine macrophages and the ability of macrophages to deacylate LPS was not diminished in endotoxin-tolerized macrophages. LPS-stimulated AOAH mRNA expression was cycloheximide sensitive, indicating that de novo protein synthesis is required for AOAH mRNA production. Moreover, AOAH mRNA expression was also induced by IFN-γ. LPS-stimulated mRNA expression was not suppressed by either dexamethasone or IL-10. Finally, intraperitoneal challenge of mice with 25 μg of LPS resulted in increases in AOAH mRNA in both the lung (∼3-fold) and in the liver (∼6-fold). A possible role for LPS-inducible AOAH in the elimination of LPS is discussed.

Regulation of gene expression and nitric oxide production in murine macrophages by the serine/threonine phosphatase inhibitor okadaic acid

LPS-stimulated macrophages produce cytokines which, at appropriate levels, direct successful immune responses against harmful pathogens. However, excessive cytokine production, as seen in endotoxemia, results in pathophysiological damage to the host. Therefore, understanding mechanisms of cytokine regulation may aid the development of strategies designed to control cytokine production during an ongoing immune response. We have examined the role of okadaic acid-sensitive phosphatases in the production of cytokines and nitric oxide by macrophages. Okadaic acid induces TNFα, IL-1β, IL-6, IFN-β, and IP-10, but not IL-10 or IL-12 (p40) mRNA. Okadaic acid differentially regulates the expression of LPS-inducible IL-10 and IL-12 (p40) mRNA. These findings suggest that okadaic acid-sensitive phosphatases are key regulators of cytokine production in unstimulated and immune-activated macrophages. Finally, okadaic acid inhibits iNOS mRNA and nitric oxide production by macrophages activated by LPS and IFN-γ.