TLR9 agonist treatment improves the therapeutic ratio of whole abdominal RT for murine colorectal tumors.

<p>Effect of TLR9 agonist treatment on tumor growth rate of BALB/c mice (n = 5) irradiated with 14 Gy AIR. Significant delay in tumor growth (p<0.0001) was observed in AIR+TLR9 agonist groups (A) with improvement of survival of the mice (p<0.008) (B) compared to AIR alone and untreated mice.</p

Xylose absorption assay.

<p>Mice treated with TLR9 agonist+WBI demonstrated significant recovery of xylose absorption post-WBI (p<0.0007 at 7 days), when compared to WBI controls. Animals treated with inhibitory Myd88 peptide, iMyd88 and WBI+TLR9 agonist failed to absorb xylose, indicating that TLR signaling was critical for the functional regeneration of intestine after radiation injury.</p

TLR9 agonist ameliorates RIGS.

<p><b>A.</b> Representative (n = 3) histology from paraffin-embedded mid-jejunal sections of mice treated with TLR9 agonist+WBI versus WBI alone (20×). <b>Hematoxylin and Eosin staining</b> demonstrates larger crypt depth and elongated villi in the mid-jejunum of mice treated with TLR9 agonist and WBI, compared to mice treated with WBI. <b>BrdU immunohistochemistry</b> demonstrates increase in crypt cell proliferation after TLR9 agonist+WBI treatment, compared to WBI controls. Note an increase in BrdU-positive crypt cells in WBI-treated mice, which is further stimulated by TLR9 agonist treatment, 3.5 days after WBI. This is a physiological response of crypt survival after WBI. <b>TUNEL staining</b> demonstrates a decrease in TUNEL-positive, apoptotic cells in TLR9 agonist+WBI mice, when compared to WBI mice. <b>B–D. </b><b>Bar graph of crypt depth, proliferation and apoptosis of crypt epithelial cells in mice treated with TLR9 agonist+WBI versus WBI alone.</b> Effect of TLR9 agonist on intestinal crypt depth (<b>B</b>), crypt proliferation rate (<b>C</b>) and tunnel positive apoptotic cells (<b>D</b>) at 1day and 3.5day post WBI. Mice treated with TLR9 agonist showed a significant resistance to radiation-induced apoptosis (p<0.001) at 1day post WBI with the increase in proliferation rate (p<0.002) and crypt depth at 3.5day post WBI (p<0.001), compared to irradiated control.</p

TLR9 agonist-activated macrophage mitigates RIGS.

<p><b>A.</b> Macrophage cell line J774A.1 was incubated with TLR9 agonist in vitro for 24 hrs. BALB/c mice transplanted with syngeneic TLR9 agonist-activated J774A.1 macrophages, 1 hr after 9.4 Gy WBI, demonstrated significant improvement in survival, compared to WBI (p<0.002) or WBI+J774A.1 (p<0.005) cohorts. <b>B. Culture supernatant of TLR9 agonist-activated J774A.1 induces clonogenic survival of rat intestinal IEC6 following irradiation.</b> Incubation of IEC6 cells with culture supernatants from TLR9 agonist-activated J774A.1 cells followed by a clonogenic assay showed an increase in surviving fraction against incremental doses of radiation (2–6 Gy) compared to irradiated control.</p

TLR9 agonist treatment increase the number of macrophages in pericryptal and lamina propria of intestine of mice treated with WBI.

<p>A. F480 Immunhistochemistry and confocal microscopic analysis and B. Quantification of number of intestinal macrophages. The number of F480+ve macrophages (indicated with arrow) increased at 1 day and 3.5 d post-WBI in the WBI+TLR9 agonist treatment group, compared to the WBI cohort. Nucleus was stained with DAPI (pseudo colored with red). Confocal microscopic images (40×) were magnified 2.3× (inset).</p

Xylose absorption assay.

<p>A time course study (1–10dys) showed significant recovery (p<0.002) of xylose absorption at 3.5 to 7 days in AdRspo1-treated cohorts, when compared to AdLacZ controls, thereby indicating the functional regeneration of intestine after radiation injury. AdLacZ-treated animals were incapable of demonstrating adequate xylose absorption after radiation injury, further contributing to animal mortality.</p

AdRspo1 treatment has no effect on tumor growth.

<p>Effect of AdRspo1 and AdLacZ treatment on tumor growth rate of Balb/c mice (n = 5) irradiated with 14Gy ABI. Significant delay in tumor growth (p<0.0001) was observed in ABI groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008014#pone-0008014-g003" target="_blank">Fig A</a>) compared to untreated mice.</p

AdRspo1 treatment induces β-catenin activation in irradiated crypts.

<p>Representative immunoblot (<b>Fig. 7A</b>) and densitometric analysis (<b>Fig. 7B</b>) of nuclear/cytosolic ratios of β-catenin from AdRspo1 and AdLacZ treated cohorts after WBI(10.4Gy). Nuclear fraction purity was validated by the absence of β-tubulin, while the purity of the cytosolic fraction was evaluated by the absence of PCNA (Fig. 7A). A continuous decline in nucear/cytosolic ratios of β-catenin was predominate in samples from irradiated AdLacZ cohorts. This is further supported by the densitometric analysis of β-catenin expression (Fig. 7B) from the nuclear/cytosolic ratio demonstrating the significant differences in AdRspo1 when compared to AdLacZ treated mice prior to (Day –1) until Day +5 post WBI.</p

AdRspo1 increases the number of regenerative crypts in irradiated mice.

<p>Effect of AdRspo1 and AdLacZ treatment on intestinal crypt depth (A), proliferation rate (B), apoptotic cells (C) at 1day and 3.5 days after WBI and the number of regenerative crypts (D) at 3.5 days after WBI. A representative sampling of thirty crypts was assessed for each treatment group.</p

Histolological assessment of intestine after Irradiation.

<p>H&E staining demonstrates increased crypt depth and increased villi thickness in AdRspo1-treated animals following exposure to WBI. BrdU immunohistochemistry demonstrates higher crypt cell proliferation after AdRspo1 treatment when compared to AdLacZ cohorts. Finally, TUNEL staining demonstrates a decrease in the rate of TUNEL-positive, apoptotic cells in AdRspo1-treated mice post-WBI, when compared to intestinal lumen of AdLacZ-treated mice.</p