Full Spectrum of LPS Activation in Alveolar Macrophages of Healthy Volunteers by Whole Transcriptomic Profiling.

Despite recent advances in understanding macrophage activation, little is known regarding how human alveolar macrophages in health calibrate its transcriptional response to canonical TLR4 activation. In this study, we examined the full spectrum of LPS activation and determined whether the transcriptomic profile of human alveolar macrophages is distinguished by a TIR-domain-containing adapter-inducing interferon-β (TRIF)-dominant type I interferon signature. Bronchoalveolar lavage macrophages were obtained from healthy volunteers, stimulated in the presence or absence of ultrapure LPS in vitro, and whole transcriptomic profiling was performed by RNA sequencing (RNA-Seq). LPS induced a robust type I interferon transcriptional response and Ingenuity Pathway Analysis predicted interferon regulatory factor (IRF)7 as the top upstream regulator of 89 known gene targets. Ubiquitin-specific peptidase (USP)-18, a negative regulator of interferon α/β responses, was among the top up-regulated genes in addition to IL10 and USP41, a novel gene with no known biological function but with high sequence homology to USP18. We determined whether IRF-7 and USP-18 can influence downstream macrophage effector cytokine production such as IL-10. We show that IRF-7 siRNA knockdown enhanced LPS-induced IL-10 production in human monocyte-derived macrophages, and USP-18 overexpression attenuated LPS-induced production of IL-10 in RAW264.7 cells. Quantitative PCR confirmed upregulation of USP18, USP41, IL10, and IRF7. An independent cohort confirmed LPS induction of USP41 and IL10 genes. These results suggest that IRF-7 and predicted downstream target USP18, both elements of a type I interferon gene signature identified by RNA-Seq, may serve to fine-tune early cytokine response by calibrating IL-10 production in human alveolar macrophages

IRF-7 is predicted as a top upstream regulator of 89 known gene targets.

<p>Analysis of RNA-Seq dataset was performed with Ingenuity Pathway Analysis software, which predicted IRF-7 to be the top upstream regulator (overlap <i>p-value</i> = 3.5x10^-37, Fisher’s Exact test, z-score 9.28, n = 5). The increased expression of <i>IRF7</i> is consistent with the increased expression state of 89 known target genes, predicted from the literature. Red lines connecting the upstream regulator and the target gene represent a positive consistent correlation. A red colored molecule represents the gene is upregulated in the dataset. More intense red color represents higher expression level state in the dataset.</p

Gene Ontology and KEGG report of down-regulated gene pathways.

<p>Gene Ontology and KEGG report of down-regulated gene pathways.</p

Gene ontology and KEGG reports of up-regulated gene pathways.

<p>Gene ontology and KEGG reports of up-regulated gene pathways.</p

Validation of RNA-sequencing data by quantitative real-time PCR.

<p>Representative up-regulated and down-regulated target genes from the RNA-Seq data set were validated with real time quantitative PCR using the same samples used for RNA-Seq assays (*<i>p</i><0.05 two-tailed t-test, vs. respective control, n = 4–5). Values represent mean (+/-) SEM of relative gene expression changes vs. 18S housekeeping gene, calculated by the ΔΔCt method. Human alveolar macrophages were stimulated in the presence or absence of LPS for 6 h.</p

USP-18 overexpression in RAW264.7 cells decreases production of IL-27 and IL-10.

<p>RAW264.7 cells were transfected with empty vector or USP-18-V5 (2 μg) for 48 hours. The cells were then incubated in the presence of LPS (200 ng/ml) or vehicle for 24 hours. Cell supernatants were assessed using ELISA showing (A) decrease in production of mouse IL-27 in USP-18-V5 transfected cells compared to empty vector in the presence of LPS and (B) decreased levels of mouse IL-10 in USP-18-V5 transfected cells after LPS stimulation. RNA was extracted from the RAW264.7 transfected cells and (C) real time quantitative PCR was performed showing decreased IL-10 RNA levels in the USP-18-V5 transfected cells compared to control vector in the presence of LPS. (D) Immunoblotting was performed in the RAW264.7 cells transfected with empty vector or USP-18-V5 (expected band size ~45 kDa) and assayed for V5 tag protein amount. β-actin detection was used as loading control.</p

Expression profile of representative genes was validated in a second, independent cohort of BAL macrophages by real-time quantitative PCR.

<p>(*<i>p</i><0.05 two-tailed paired t-test vs. respective control, n = 24). Values represent mean (+/-) SEM of relative gene expression changes vs. 18S housekeeping gene, calculated by the ΔΔCt method. Human alveolar macrophages in the second, independent cohort were stimulated in the presence or absence of LPS for 2 h.</p

<i>IRF7</i> gene knockdown enhances LPS-induced human macrophage IL-10 production.

<p>Human monocyte derived macrophages were isolated from the peripheral blood of healthy volunteers and cultured for 5 days in RPMI 1640 media containing fresh media with 10% autologous serum and recombinant human-M-CSF (50 ng/ml) before transfection with control siRNA or IRF-7 siRNA. The cells were stimulated in the presence or absence of ultrapure LPS (100 ng/ml, <i>E</i>. <i>coli</i> 0111:B4) for 24 hours. (A) RT-PCR for IRF-7 was performed and confirmed gene knockdown with IRF-7 specific pooled siRNA as compared to control siRNA-treated cells after exposure to LPS (expected amplicon size IRF-7: 327 bp). β-actin gene was used as a house-keeping gene controlling for input sample quantity (expected amplicon size β-actin: 353 bp). (B) IRF-7 siRNA treated cells showed attenuated IRF-7 protein expression by immunoblotting, when compared to control siRNA treated cells in the presence of LPS (expected band size IRF-7 ~50 kDa). Alpha tubulin was used as loading control. Unstimulated RAW264.7 cell lysates were used as positive control for IRF-7. (C) Cell culture supernatants from HMDM were harvested for quantification of IL-10 production by ELISA. IRF-7 knockdown enhances IL-10 production in the presence of LPS. IL-10 production is minimal in the absence of LPS. (D) Composite of 8 independent experiments showing significant increase in IL-10 production by ELISA in supernatants of IRF-7 siRNA treated HMDMs compared to control siRNA in the presence of LPS. Each point represents matched samples, (*<i>p-value</i> < 0.05 Wilcoxon matched-pairs sign rank test, n = 8).</p