Genes that exhibit the greatest expression deficit in <i>Lm</i>-stimulated cord blood monocytes in comparison to adult monocytes are regulated by IRF3 and/or Type I IFNs.

<p>(A) <i>Lm</i>-induced genes exhibiting statistically significant differential expression in neonates and young adults (n = 123) were grouped according to the time point at which their maximum transcript levels were observed (2 or 6 hrs). They were then grouped according to their relative maximum transcript levels in cord blood (neonates) versus young adults. Induced genes with a higher maximum transcript level in neonates than young adults (with statistically significant differential expression) are included in classes I (2-hr peak) and V (6-hr peak) (column 7). Genes exhibiting a maximum transcript level in neonates that was 50–100% of the young adult transcript level (but with statistically significant differential expression) are included in classes II (2-hr) and VI (6-hr). Genes exhibiting a maximum transcript level in neonates that was 20–50% of the young adult transcript level are in classes III (2-hr) and VII (6-hr). Genes with a maximum transcript level in neonates below 20% of the young adult transcript level are in classes IV (2-hr) and VIII (6-hr). Columns 1–6 show the relative transcript levels (based on the log-transformed mean-centered RPKM) for these 123 classified genes in all samples and all time points from both neonates and young adults. Column 8 indicates genes that lack obvious mouse orthologs (lightest pink), genes that contain mouse orthologs that are either not expressed or not induced in mouse bone marrow-derived macrophages (dark pink), and genes containing mouse orthologs that are both expressed and induced by LPS (red). Columns 9 and 10 show relative expression of the mouse ortholog of the human gene in Lipid A-stimulated macrophages from IRF3^-/- and IFNAR^-/- mice, respectively (see blue scale at right). Note that these columns are only relevant for genes shown in red in Column 8. Column 11 indicates genes with promoters that contain an IRF1 transcription factor binding motif between -450 and +50 bps relative to the transcription start site. (B) Enrichment of transcription factor binding sites determined using the Pscan program is shown for each gene class from panel A. Color intensity is proportional to the negative log(p-value).</p

Hierarchical clustering of LPS-stimulated monocyte transcriptomes from human neonates, adults, and older adults.

<p>(A) RNA-seq experiments were performed with three independent human monocyte samples from cord blood (N), young adult peripheral blood (A), and older adult peripheral blood (OA) stimulated with LPS for 0, 1, and 6 hrs. Hierarchical clustering was performed with the 1147 genes found to be induced by at least 5-fold at the 1- or 6-hr time point in at least one sample and with an induced RPKM of at least 4 (genes smaller than 200 bp were also excluded from the analysis). Sample codes correspond to the age abbreviation followed by the sample number (1 through 3 for each age); the time point (0, 1, or 6 hr) is indicated after the period. Inducible transcriptomes exhibit strong time-dependent clustering, with much less age-dependent clustering. (B) Pearson correlation values (R) used for the hierarchical clustering in panel A are shown. Each time point from each sample was compared to every other sample and time point. R values are color-coded from low (green) to high (red). Samples on the X and Y axes are grouped first according to age group, then time point (0, 1, or 6), and then sample number (1–3).</p

Analysis of <i>Lm</i>-induced genes in monocytes by K-means cluster analysis.

<p>(A) The 865 genes that exceeded 200 bp in length, exhibited an RPKM of at least 4 in one sample, and were induced by <i>Lm</i> infection by at least 5-fold in the same sample were divided into 10 clusters by k-means cluster analysis, which considers similarities in transcript levels for each gene across all 27 samples (3 age groups, 3 samples for each age group, and 3 time points for each sample). The three independent samples are shown in parallel for each age group. Colors indicate the percentile of the relative expression level (based on the log-transformed mean-centered RPKM for each gene), as indicated at the bottom. (B) The average relative transcript levels for genes within each cluster and are shown for each age group (neonates, blue diamonds; young adults, red squares; older adults, green triangles).</p

Genes that exhibit the greatest expression deficit in LPS-stimulated cord blood monocytes in comparison to adult monocytes are regulated by IRF3 and/or Type I IFNs.

<p>(A) LPS-induced genes exhibiting statistically significant differential expression in neonates and adults (n = 118) were grouped according to the time point at which their maximum transcript levels were observed (1 or 6 hrs). They were then grouped according to their relative maximum transcript levels in cord blood (neonates) versus young adults. Induced genes with a higher maximum transcript level in neonates than young adults (with statistically significant differential expression) are included in classes I (1-hr peak) and III (6-hr peak) (column 7). Genes exhibiting a maximum transcript level in neonates that was 50–100% of the young adult transcript level (but with statistically significant differential expression) are included in class IV (no genes with peak transcript levels at 1-hr fit this criterion). Genes exhibiting a maximum transcript level in neonates that was 20–50% of the young adult transcript level are in classes II (1-hr) and V (6-hr). Genes with a maximum transcript level in neonates below 20% of the young adult transcript level are in class VI. The differential expression of six of these genes was confirmed by quantitative RT-PCR (data not shown). Columns 1–6 show the relative transcript levels (based on the log-transformed mean-centered RPKM) for these 118 classified genes in all samples and all time points from both neonates and young adults. Column 8 indicates genes that lack obvious mouse orthologs (lightest pink), genes that contain mouse orthologs that are either not expressed or not induced in mouse bone marrow-derived macrophages (dark pink), and genes containing mouse orthologs that are both expressed and induced by LPS (red). Columns 9 and 10 show relative expression of the mouse ortholog of the human gene in Lipid A-stimulated macrophages from IRF3^-/- and IFNAR^-/- mice, respectively (see blue scale at right). Note that these columns are only relevant for genes shown in red in Column 8. Column 11 indicates genes with promoters that contain an IRF1 transcription factor binding motif between -450 and +50 bps relative to the transcription start site. (B) Enrichment of transcription factor binding sites determined using the Pscan program is shown for each gene class from panel A. Color intensity is proportional to the negative log(p-value).</p

Elevated expression of a broad range of inflammatory genes prior to stimulation of freshly isolated monocytes from older adults.

<p>(A) LPS-induced genes exhibiting differential basal expression between adults and older adults (n = 189) are grouped according to maximum mRNA level. Columns 7 and 8 show the ratio of transcript levels between older adults and young adults before stimulation and at maximum transcript levels, respectively. (B) The average relative transcript levels within each cluster and for each age are shown.</p

Hierarchical clustering of <i>Lm</i>-infected monocyte transcriptomes from human neonates, adults, and older adults.

<p>(A) RNA-seq experiments were performed with three independent human monocyte samples from cord blood (N), young adult peripheral blood (A), and older adult peripheral blood (OA) infected with <i>Lm</i> for 0, 2, and 6 hrs. Hierarchical clustering was performed with the 865 genes found to be induced by at least 5-fold at the 2- or 6-hr time point in at least one sample and with an induced RPKM of at least 4 (genes smaller than 200 bp were also excluded from the analysis). Sample codes correspond to the age abbreviation followed by the sample number (1 through 3 for each age); the time point (0, 2, or 6 hr) is indicated after the period. Inducible transcriptomes exhibit strong time-dependent clustering, with much less age-dependent clustering. (B) Pearson correlation values (R) used for the hierarchical clustering in panel A are shown. Each time point from each sample was compared to every other sample and time point. R values are color-coded from low (green) to high (red). Samples on the X and Y axes are grouped first according to age group, then time point (0, 2, or 6), and then sample number (1–3).</p

Analysis of LPS-induced genes in monocytes by K-means cluster analysis.

<p>(A) The 1147 genes that exceeded 200 bp in length, exhibited an RPKM of at least 4 in one sample, and were induced by LPS by at least 5-fold in the same sample were divided into 10 clusters by k-means cluster analysis, which considers similarities in transcript levels for each gene across all 27 samples (3 age groups, 3 samples for each age group, and 3 time points for each sample). The three independent samples are shown in parallel for each age group. Colors indicate the percentile of the relative expression level (based on the log-transformed mean-centered RPKM for each gene), as indicated at the bottom. (B) The average relative transcript levels for genes within each cluster are shown for each age group (neonates, blue diamonds; young adults, red squares; older adults, green triangles).</p

Amino Acid-Dependent Attenuation of Toll-like Receptor Signaling by Peptide-Gold Nanoparticle Hybrids

Manipulation of immune responsiveness using nanodevices provides a potential approach to treat human diseases. Toll-like receptor (TLR) signaling plays a central role in the pathophysiology of many acute and chronic human inflammatory diseases, and pharmacological regulation of TLR responses is anticipated to be beneficial in many of these inflammatory conditions. Here we describe the discovery of a unique class of peptide-gold nanoparticle hybrids that exhibit a broad inhibitory activity on TLR signaling, inhibiting signaling through TLRs 2, 3, 4, and 5. As exemplified using TLR4, the nanoparticles were found to inhibit both arms of TLR4 signaling cascade triggered by the prototypical ligand, lipopolysaccharide (LPS). Through structure–activity relationship studies, we identified the key chemical components of the hybrids that contribute to their immunomodulatory activity. Specifically, the hydrophobicity and aromatic ring structure of the amino acids on the peptides were essential for modulating TLR4 responses. This work enhances our fundamental understanding of the role of nanoparticle surface chemistry in regulating innate immune signaling, and identifies specific nanoparticle hybrids that may represent a unique class of anti-inflammatory therapeutics for human inflammatory diseases

Humanized TLR4/MD-2 Mice Reveal LPS Recognition Differentially Impacts Susceptibility to <em>Yersinia pestis</em> and <em>Salmonella enterica</em>

<div><p>Although lipopolysaccharide (LPS) stimulation through the Toll-like receptor (TLR)-4/MD-2 receptor complex activates host defense against Gram-negative bacterial pathogens, how species-specific differences in LPS recognition impact host defense remains undefined. Herein, we establish how temperature dependent shifts in the lipid A of <em>Yersinia pestis</em> LPS that differentially impact recognition by mouse versus human TLR4/MD-2 dictate infection susceptibility. When grown at 37°C, <em>Y. pestis</em> LPS is hypo-acylated and less stimulatory to human compared with murine TLR4/MD-2. By contrast, when grown at reduced temperatures, <em>Y. pestis</em> LPS is more acylated, and stimulates cells equally via human and mouse TLR4/MD-2. To investigate how these temperature dependent shifts in LPS impact infection susceptibility, transgenic mice expressing human rather than mouse TLR4/MD-2 were generated. We found the increased susceptibility to <em>Y. pestis</em> for “humanized” TLR4/MD-2 mice directly paralleled blunted inflammatory cytokine production in response to stimulation with purified LPS. By contrast, for other Gram-negative pathogens with highly acylated lipid A including <em>Salmonella enterica</em> or <em>Escherichia coli</em>, infection susceptibility and the response after stimulation with LPS were indistinguishable between mice expressing human or mouse TLR4/MD-2. Thus, <em>Y. pestis</em> exploits temperature-dependent shifts in LPS acylation to selectively evade recognition by human TLR4/MD-2 uncovered with “humanized” TLR4/MD-2 transgenic mice.</p> </div

Geometric mean ratio (GMR) and 95% CI of <i>in vitro</i> cytokine concentrations in the unstimulated RPMI and Lyovec™ wells of 52 Kawasaki disease (KD), 20 toxic shock syndrome (TSS) and 53 control participants.

<p>Abbreviations: IFN, interferon; TNF, tumour necrosis factor; IL, interleukin; IL-1ra, receptor antagonist.</p