Expression level of human TLR4 rather than sequence is the key determinant of LPS responsiveness

<div><p>To address the role of Toll-like receptor 4 (TLR4) single nucleotide polymorphisms (SNP) in lipopolysaccharide (LPS) recognition, we generated mice that differed only in the sequence of TLR4. We used a bacterial artificial chromosome (BAC) transgenic approach and TLR4/MD-2 knockout mice to specifically examine the role of human TLR4 variants in recognition of LPS. Using <i>in vitro</i> and <i>in vivo</i> assays we found that the expression level rather than the sequence of TLR4 played a larger role in recognition of LPS, especially hypoacylated LPS.</p></div

Cytokine responses in supernatants from BMDM stimulated with indicated LPS.

<p>An equal number (10⁶ cells) of BMDM were plated in 96-well plates and stimulated with 1000 ng/ml PA or YP LPS, or 10 ng/ml EC LPS. Cytokines secreted into the supernatants were measured 24-hr later by Luminex. Results from 2 independent BMDM preparations are shown. * above genotypes shows results compared to B6 whereas † is compared to KO. 1-way ANOVA results for the EC IL-6 responses are shown in the table.</p

TLR4 expression correlates with copy number.

<p>(A) BMDM from each of the genotypes indicated above plots were stained with anti-huTLR4 clone HTA125 or isotype control. Boxed number in each plot shows ΔMFI of TLR4 (red histogram for huTLR4, blue histogram in B for muTLR4) vs. isotype control (filled grey histogram). (B) B6 mouse BMDM stained with anti-muTLR4. (C) Each symbol represents a separate BMDM preparation. All cells express huMD-2; red squares depict huTLR4^WT, light blue triangles huTLR4^D299G (299), and dark blue inverted triangles huTLR4^D299G+T399I (399). X (KO) does not express TLR4. Open symbols have lower copy numbers than closed symbols. Brackets show significant pair-wise comparisons using 1-way ANOVA followed by Bonferroni’s Multiple Comparison test. (D) Relative TLR4 mRNA expression in BMDM from each genotype, including WT B6 BMDM (average of 3 separate BMDM preparations). The same primers were used for human and mouse TLR4, and expression relative to ß-actin is shown. Plotted are the means +/- SD. 1-way ANOVA results for D are shown to the right. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001, ns = not significant.</p

Primer sequences.

<p>Primer sequences.</p

Serum cytokine responses to 50 μg PA LPS 1 + 6 hr post IP injection.

<p>Mice of the indicated genotypes were injected IP with PA LPS and then bled from the retro-orbital sinus 1 hr after injection. At 6 hr, the mice were euthanized for a terminal sample collection. Shown are combined data from 2 experiments. Data are plotted on a log scale for easier visualization due to large variation. * above symbols shows results for that genotype compared to B6 whereas † is compared to KO.</p

All TLR4 SNP primary splenocyte macrophage/monocyte populations respond to EC LPS as measured by intracellular cytokine staining.

<p>(A) Percent of mac/mono cells that are TNF+ in response to 1000 ng/ml EC LPS or (B) 10 μg/ml CpG. (C) Percent of mac/mono TNF+ in response to EC LPS from (A) divided by the percent TNF+ in response to CpG from (B). † above genotype show results compared to KO. † <i>P</i><0.05, †† <i>P</i><0.01, ††† <i>P</i><0.001, †††† <i>P</i><0.0001, ns = not significant. Brackets show significant pairwise comparisons. Combined data from 7 experiments with each symbol representing a separate experiment for each genotype. Note that not each genotype was tested in each experiment. huWT = huTLR4^WT, D299G = huTLR4^D299G, T399I = huTLR4^D299G+T399I, number after dash shows copy number of huTLR4 transgene.</p

Serum cytokine responses to 100 μg EC LPS 1 + 6 hr post IP injection.

<p>Mice of the indicated genotypes were injected IP with EC LPS and then bled from the retro-orbital sinus 1 hr after injection. At 6 hr, the mice were euthanized for a terminal sample collection. Shown are combined data from 3 experiments, with 1 experiment containing 1 and 6 hr time points whereas the other 2 had either the 1 or 6 hr time point. All genotypes were included in all experiments. Data are plotted on a log scale for easier visualization due to large variation. Line indicates mean for genotype. * above symbols shows results compared to B6 whereas † is compared to KO.</p

Reduced responsiveness to hypoacylated LPS by TLR4 SNP primary splenocyte mac/mono populations.

<p>Percent of mac/mono cells that are TNF+ in response to 1000 ng/ml PA LPS, YP LPS or MPL, top row, or percent TNF+ corrected by the percent TNF+ in response to CpG, bottom row. * above genotypes shows results compared to B6 whereas † is compared to KO. Data for huTLR4^WT in response to MPL were combined for 1-way ANOVA since the 4-copy line was only tested once and our previous studies did not reveal any differences between the 2- and 4-copy lines.</p

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

Decreased responsiveness to LPS of <i>Y. pestis</i> grown at 37°C by huTLR4/MD-2 <i>in vitro</i>.

<p>HEK-293 cells expressing the indicated proteins were stimulated for 4 hr with increasing concentrations of <i>E. coli</i> (EC, blue), <i>P. aeruginosa</i> (PA), or <i>Y. pestis</i> (YP, red, grown at 37°C) LPS or Lipid IVa. Firefly luciferase was induced by NF-kB activation (ELAM promoter) and corrected by the constitutively active ß-actin driven Renilla luciferase. Relative Light Units plotted are the direct Firefly/Renilla values. Shown is the average (+/−SD) of triplicate wells from one representative experiment of 2. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002963#ppat.1002963.s006" target="_blank">Table S1</a> for statistical analysis.</p