IL-10 Knockout Reporter MØ Fail to Down Regulate Class II in Response to FTMØSN.

<p>FTMØSN from wild type (WT) and IL-10Δ BMMØ was used to treat either WT (upper two panels) or IL-10Δ (lower two panels) reporter MØ. Reporter MØ were also treated with either media alone or supernatant from non-infected MØ (Mock). After overnight culture, the levels of class II (II) and GAPDH (G) was monitored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037330#pone-0037330-g004" target="_blank">Figure 4</a>. Shown are representative results from 1 of 3 independent experiments.</p

MARCH1Δ and Class II K225R BMMØ Fail to Down-Regulate Class II in Response to FTMØSN.

<p>IFN-γ activated wild type (WT), MARCH1Δ (M1Δ) or class II K225R (K>R) BMMØ were treated with mock SN or FTMØSN. After 20–24 hours, total MØ class II (II) and GAPDH (G) levels were determined by western blot analysis of whole cell lysates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037330#pone.0037330-Wilson1" target="_blank">[7]</a>. Shown are representative results from 1 of 3 independent experiments.</p

FTMØSN Production and Action.

<p><i>F. tularensis</i> infected “producer” MØ make PGE₂, which acts in an autocrine/paracrine fashion to drive the production of FTMØSN (<i><u>F</u>. <u>t</u>ularensis </i><u>MØ s</u>uper<u>n</u>atant). FTMØSN contains a soluble factor of >10 kDa molecular mass that elicits the ubiquitin-dependent down-regulation of “reporter” MØ MHC class II molecules <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037330#pone.0037330-Wilson1" target="_blank">[7]</a>. In this report, we establish that the active factor in FTMØSN is distinct from IL-10, but that “reporter” MØ class II down-regulation is driven by induction of <i>reporter</i> MØ IL-10 production, which drives MARCH1 expression and class II ubiquitination. This means that FTMØSN contains a factor distinct from IL-10, which is induced by <i>F. tularensis</i>-elicited PGE₂ and which is able to drive reporter MØ IL-10 production.</p

Recombinant IL-10 Down-regulates Reporter MØ Class II Expression.

<p>Reporter MØ were treated with FTMØSN or 100 ng/ml of recombinant IL-10 (rIL-10). After overnight culture, the levels of class II (II) and GAPDH (G) was monitored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037330#pone-0037330-g004" target="_blank">Figure 4</a>. Shown are representative results from 1 of 3 independent experiments.</p

Expression of SNAP-23 protein is reduced by half in <i>Snap23</i>^fl/wt and <i>Snap23</i>^Δ/wt mice.

<p>(<b>A</b>) <i>Snap23</i>^fl/wt heterozygous mice were mated and twelve two-week old pups from this mating were genotyped and analyzed for SNAP-23 protein expression. Genotyping of tail DNA was performed using PCR Primer set 2 from tail DNA. A single small PCR fragment (266 bp) is present in <i>Snap23</i>^wt/wt pups, and double fragments (266 and 400 bp) is present in <i>Snap23</i>^fl/wt pups. No homozygous <i>Snap23</i>^fl/fl pups were obtained when more than 50 pups were analyzed from <i>Snap23</i>^fl/wt heterozygous matings. For immunoblot analysis, whole brain was solubilized in modified RIPA lysis buffer and protein levels were analyzed by immunoblotting (WB) as indicated antibodies. (<b>B</b>) <i>Snap23</i>^Δ/wt heterozygous mice were mated and pups from this mating were genotyped and their brains were analyzed for expression of SNAP-23 and other SNARE proteins. Genotyping of tail DNA was performed using PCR Primer set 2. A single small PCR fragment (266 bp) is present in <i>Snap23</i>^wt/wt pups, and double fragments (266 and 492 bp) is present in <i>Snap23</i>^Δ/wt pups. No homozygous <i>Snap23</i>^Δ/Δ pups were ever obtained from <i>Snap23</i>^Δ/wt heterozygous matings. Whole brains were solubilized and analyzed by immunoblotting (WB) using the indicated antibodies.</p

<i>Snap23</i>^Δ/Δ blastocysts die prior to uterine implantation.

<p>(<b>A</b>) To evaluate the timing of embryonic lethality, embryos were collected from super-ovulated <i>Snap23</i>^Δ/wt females mated with <i>Snap23</i>^Δ/wt male mice by uterine flushing at E3.5. About 1/4 of the isolated blastocysts were morphologically abnormal and appeared to be degenerating; unlike sibling normal blastocysts they failed to develop any further during 24 hrs of culture (indicated by red arrows; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018444#pone-0018444-t002" target="_blank">Table 2</a>). (<b>B</b>) Representative example of genotyping analysis revealing that abnormal blastocysts are homozygous for the <i>Snap23</i> deleted allele (<i>Snap23</i>^Δ/Δ). Genomic DNA was isolated from individual blastocysts (shown in panel (A)) following 24 hr in culture, and genotyping was conducted using primers genoE2 SS, genoE2 AS, and genoE3 rev. PCR products for the <i>Snap23</i>^wt allele (266 bp) and for the <i>Snap23</i>^Δ allele (492 bp) are indicated.</p

Blocking the IL-10 Receptor Blocks FTMØSN-induced Reporter MØ Class II Down-regulation.

<p>Reporter MØ were treated with 20 mg/ml anti-IL-10R blocking mAb for 20 min. at 37uC or left untreated before addition of an equal volume of undiluted FTMØSN. After overnight culture, the levels of class II (II) and GAPDH (G) was monitored as in Figure 4. Shown are representative results from 1 of 3 independent experiments.</p

Genotype analysis from timed-pregnant matings of <i>Snap23</i>^Δ/wt heterozygote mice.

<p>Embryo age, number of dissected embryos, and <i>Snap23</i> genotype results are summarized as indicated.</p

Oligonucleotide primers used in this study and estimated size of PCR products.

<p>The oligonucleotide sequences used in this study are listed in the upper table. The oligonucleotide primers were used to genotype the mice, to obtain genomic fragments of <i>Snap23</i>, and to generate probes for Southern blot analysis. The estimated sizes of PCR products obtained during genotyping the mice are indicated in the lower table.</p