Lipopolysaccharide Stimulates p38-dependent Induction of Antiviral Genes in Neutrophils Independently of Paracrine Factors

Abstract

Lipopolysaccharide (LPS) induces neutrophils to synthesize and secrete pro-inflammatory cytokines and chemokines, which are regulated at both the transcriptional and translational level. We reported previously that neutrophils stimulated with LPS induce expression of genes typically expressed in response to stimulation with antiviral type I interferons (IFN), such as myxovirus resistance-1 (MX1). However, we present evidence that this response of neutrophils to lipopolysaccharide occurs in the absence of interferon-dependent signaling. Lipopolysaccharide-stimulated neutrophils do not phosphorylate the interferon-associated transcription factors signal transducer and activator of transcription-1 and -3, and medium from lipopolysaccharide-stimulated cells was unable to induce MX1 gene expression, suggesting a soluble factor is not involved. Furthermore, LPS did not alter expression of IFNA and IFNB genes. In contrast to neutrophils, LPS-stimulated human monocyte-derived macrophages induced the expression of MX1, but IFNB was induced, and medium from LPSstimulated monocyte-derived macrophages supported MX1 induction. An inhibitor of p38 kinase blocked induction of MX1 by lipopolysaccharide, but not IFN␣, in neutrophils, and induction of MX1 was dependent on protein synthesis. LPS, but not IFN␣, substantially activated p38. In contrast, the induction of MX1 by LPS in monocyte-derived macrophages was insensitive to p38 inhibition, although p38 is phosphorylated in LPS-stimulated but not IFN␣-stimulated monocyte-derived macrophages. The expression of MX1 in neutrophils and monocyte-derived macrophages is mediated by TLR4 but not TLR2. The data presented here indicate that lipopolysaccharide activates novel interferon-independent signaling pathways in neutrophils and that induction of antiviral genes is a consequence of exposure of neutrophils to bacterial products.

Lipopolysaccharide-Binding Protein Critically Regulates Lipopolysaccharide-Induced IFN-β Signaling Pathway in Human Monocytes

“…It is noteworthy that the induction of IFIT1 and IP-10, but not of TNF-␣ or IFN-␤, seems to require de novo protein synthesis. This fact, along with the LBP-dependent Tyk2 and STAT1 phosphorylation, is consistent with previous reports that the induction of these molecules is regulated by IFN-␤ (6,7,44).…”

“…LPS also induces large amounts of antiviral proteins (6,7) and CXCR3 chemokines (8), all of which are known to be regulated by type I or II IFNs (9). Transcriptional regulation of these cytokines and chemokines is mediated by NF-B and IFN-regulatory factor (IRF)-3 (7).…”

The MYD88-Independent Pathway Is Not Mobilized in Human Neutrophils Stimulated via TLR4

“…The reliability of our microarray neutrophil data is further illustrated by the fact that we observed no induction of either IL-6 or CCL2 signals in response to LPS, which have been shown to reflect the contamination of neutrophil RNA samples by RNA from monocytes and/or eosinophils (8,42). This possibly explains some discrepancies between the data reported herein and recent microarray data obtained from LPS-treated human neutrophils, which featured a dramatic up-regulation not only of IL-6 or CCL2 but also of many classically IFN␤-inducible mRNAs such as MX1, G1P2/ISG15, IFIT1, IFIT2, IFIT3, IFIT5, IFI35, and EIF2AK2/ PKR (69,70). In keeping with this interpretation, these earlier microarray studies used neutrophil preparations that were stated to contain a substantial proportion of contaminant cells (as high as 5%) (69,70) that may well give rise to false positives.…”

“…This possibly explains some discrepancies between the data reported herein and recent microarray data obtained from LPS-treated human neutrophils, which featured a dramatic up-regulation not only of IL-6 or CCL2 but also of many classically IFN␤-inducible mRNAs such as MX1, G1P2/ISG15, IFIT1, IFIT2, IFIT3, IFIT5, IFI35, and EIF2AK2/ PKR (69,70). In keeping with this interpretation, these earlier microarray studies used neutrophil preparations that were stated to contain a substantial proportion of contaminant cells (as high as 5%) (69,70) that may well give rise to false positives. Clearly, the purity of the cell populations will increasingly constitute an important concern in future microarray studies conducted in primary cells.…”

Shift in Monocyte Apoptosis with Increasing Viral Load and Change in Apoptosis-Related ISG/Bcl2 Family Gene Expression in Chronically HIV-1-Infected Subjects

“…Change in ISG expression and increasing sCD14 with viremia may evidence increasing microbial translocation, as microbial products were associated with interferon alpha activation in HIV-1 viremic patients (17) and may contribute to the shift in monocyte ISG expression and apoptosis induction (64,65). Previous studies have demonstrated elevated sCD14 in multiple comparisons of HIV ϩ to HIV Ϫ donors (14,16,17,(66)(67)(68), and while we demonstrated an association with viral load and CD4 count ( Fig.…”