Putative model of TLR11 ligand recognition.

<p>(A) Cathepsin-derived C-terminal region of TLR11 interacts with FliC within an acidic endolysosomal compartment. Both N- and C-terminal LRRs of TLR11 can interact with FliC independently. (B) Non-cleaved, full-length TLR11 interacts with TPRF outside the lysosomal compartment. Both the N- and C-terminal LRRs of TLR11 are required for TPRF interaction.</p

Deletion of C-terminal LRRs of TLR11-Flag impair His-TPRF interaction.

<p>(A) Schematic representation of WT or truncated-TLR11-Flag. Numbers represent the initial amino acid of deleted region or a domain. (B) His-TPRF pulldown assay at pH 6.0. HEK-293T cells were transiently transfected with C-terminal Flag-tagged TLR11 or truncated mutants. These experiments were performed like in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148987#pone.0148987.g002" target="_blank">Fig 2A</a>. Data are representative of three times experiments.</p

Cleaved TLR11-Flag strongly interacts with His-FliC but not His-TPRF.

<p>(A) Pulldown assay at pH6.0. HEK-293T cells were transiently transfected with C-terminal Flag-tagged TLR and cleared lysates were incubated with the indicated ligand at pH 6.0. His-TPRF and His-FliC were pulled down by TALON resin and anti-FliC antibody conjugated G-sepharose beads, respectively. TLR binding was analyzed by Western blots. Arrow heads indicate the position of cleaved TLR11-Flag band. Data are representative of at least 10 independent experiments; immunoprecipitation (IP). (B) Ratio of the C-terminal fragment of cleaved TLR11 to full-length TLR11 was determined by densitometric analysis of Western blots using ImageJ. Data represents the mean, with error bars indicated standard deviation, from seven independent experiments by using HEK-293T cells. *p < 0.0001, FliC IP versus TPRF IP.</p

TLR11-Flag is cleaved and its cleavage is sensitive to cathepsin inhibition.

<p>HEK-293T cells were transiently transfected with C-terminal Flag-tagged TLR11. Cells were treated with either DMSO or 20 uM z-FA-fmk (z-FA) before lysis. Lysates were analyzed by Western blot with anti-Flag antibody: full-length (FL), immunoblotting (IB), untransfected cells (UN).</p

Mutations in the N-terminal region of TLR11 impair His-TPRF binding.

<p>(A) Schematic representation of TLR11 domains: signal sequence (SS), ectodomain (EC), transmembrane domain (TM), cytoplasmic domain (Cyt). Numbers represent the putative initial amino acid of each domain. (B) His-TPRF pulldown assay at pH6.0. HEK-293T cells were transiently transfected with C-terminal Flag-tagged TLR11 or its mutants. These experiments were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148987#pone.0148987.g002" target="_blank">Fig 2A</a>. Arrow heads indicate the position of cleaved TLR11-Flag band. Data are representative of four independent experiments.</p

Acidic conditions are required for TLR11-FliC interaction.

<p>Pulldown assay at either pH 6.0 or pH 7.0, These experiments were performed like in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148987#pone.0148987.g002" target="_blank">Fig 2A</a> with changing pH condition. Data are representative of six independent experiments.</p

Keratoacanthoma development in the PD mice.

<p>Chemical carcinogenesis study in heterozygous PD and wild-type littermates. A. Papilloma formation in a PD heterozygote during TPA treatment shows no difference between experimental and control groups of mice. B. Clinical appearance of a typical papilloma. C. PD heterozygous mouse that has developed a typical keratoacanthoma showing a keratinaceous core and well-defined tumor. D. Histologic examination of keratoacanthomata from PD heterozygous mice showing classic appearance of these tumors. E. Mutational analysis of PD mice for <i>Hras</i> codons 12,13,61 and <i>Tp53</i>.</p

Generation of PD mice.

<p>Targeted knock-in at <i>RelA</i> to generate a S276D amino acid transition from exon 7 in mice creating the PD targeting vector. A. Strategy for generating the targeted knock-in including the coding change at serine 276 and addition of an EagI restriction site to allow for genotyping. B. Genotyping of the PD mouse shows a 722 base pair (bp) cDNA in the wild-type mouse that can be cut in to two pieces (356 and 366 bp) with EagI in the presence of the PD allele. C. Homozygous PD mice are born in fewer numbers than expected by normal Mendelian ratios. C. Approximately 10 day old littermates from a male and female heterozygous PD breeding. Homozygous PD mice are runted, erythrodermic, alopecic, and scaly. At this point in development, wild-type littermates demonstrate normal hair growth and size. D. Immunohistochemistry staining of mouse skin showing few F4/80+ cells in wild-type tissue (left) compared to the large infiltrate in PD homozygous mouse skin (middle). Spleen shows typical F4/80+ staining as a control (right).</p

Proliferation and Dysplasia of PD mouse skin.

<p>A. Homozygous PD mouse skin at 10 days showing focus of epidermal dysplasia with aggregates of basaloid cells in the epidermis without penetration of the basement membrane. B. Examination of proliferation of epidermal keratinocytes in mouse skin with Ki-67. Nuclear Ki-67 is restricted to the basal keratinocytes in wild-type littermate controls (top) whereas the PD homozygous mouse has positive staining at all levels of the epidermis (bottom). Skin treated with a rat isotype control shows no non-specific staining in the epidermis (not shown).</p

Flagellin and CpG ODN induce robust innate inflammatory infiltrates in the lung.

<p>(<b>A</b>), (<b>B</b>) Neutrophil accumulation in the airways one day after a single i.n. administration (d1) of OVA (O) or OVA plus flagellin (1 μg) (OFla) in wildtype, <i>Tlr5</i>^<i>-/-</i><i>Tlr11</i>^<i>-/-</i>, and <i>Nlrc4</i>^<i>-/-</i> mice (<b>A</b>) or in wildtype, <i>Tlr5</i>^<i>-/-</i><i>Tlr11</i>^<i>-/-</i> and <i>Tlr4</i>^<i>-/-</i> mice (<b>B</b>), as assessed by flow cytometry of the cells in the BAL fluid. (<b>C</b>) Cellular composition of the innate inflammatory infiltrate in the lung one day after the third i.n. sensitization (d3) with OVA, OVA plus flagellin (1 μg), or OVA plus CpG ODN (3 μg) (OCpG). Data in (<b>A</b>) contain 4 mice per group and are representative of two independent experiments, data in (<b>B</b>) contain 4 mice per group and are representative of two independent experiments, and data in (<b>C</b>) contain 3–4 mice per group and are representative of three independent experiments. Error bars indicate mean +SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 using one-way anova with Bonferroni post-test. In (<b>A</b>) and (<b>B</b>), all groups of OVA-treated mice have statistically significant different values compared to OVA plus flagellin-treated wild-type mice (<b>A</b> and <b>B</b>), <i>Nlrc4</i>^<i>-/-</i> (<b>A</b>), and <i>Tlr4</i>^<i>-/-</i> mice (<b>B</b>) (*** P ≤ 0.001 for all comparisons; not indicated on the panel).</p