TLR9 Agonist Protects Mice from Radiation-Induced Gastrointestinal Syndrome
Citation: Saha S, Bhanja P, Liu L, Alfieri AA, Yu D, et al. (
TLR9 Agonist Protects Mice from Radiation-Induced Gastrointestinal Syndrome
Subhrajit Saha 0
Payel Bhanja 0
Laibin Liu 0
Alan A. Alfieri 0
Dong Yu 0
Ekambar R. Kandimalla 0
Chandan Guha 0
Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America
0 1 Department of Radiation Oncology, Albert Einstein College of Medicine, The Montefiore Medical Center, Bronx, New York, United States of America, 2 Department of Pathology, Albert Einstein College of Medicine, The Montefiore Medical Center, Bronx, New York, United States of America, 3 Idera Pharmaceuticals, Inc. , Cambridge, Massachusetts , United States of America
Purpose: Radiation-induced gastrointestinal syndrome (RIGS) is due to the clonogenic loss of crypt cells and villi depopulation, resulting in disruption of mucosal barrier, bacterial invasion, inflammation and sepsis. Intestinal macrophages could recognize invading bacterial DNA via TLR9 receptors and transmit regenerative signals to the neighboring crypt. We therefore investigated whether systemic administration of designer TLR9 agonist could ameliorate RIGS by activating TLR9. Methods and Materials: Male C57Bl6 mice were distributed in four experimental cohorts, whole body irradiation (WBI) (8.410.4 Gy), TLR9 agonist (1 mg/kg s.c.), 1 h pre- or post-WBI and TLR9 agonist+WBI+iMyd88 (pretreatment with inhibitory peptide against Myd88). Animals were observed for survival and intestine was harvested for histological analysis. BALB/c mice with CT26 colon tumors in abdominal wall were irradiated with 14 Gy single dose of whole abdominal irradiation (AIR) for tumor growth study. Results: Mice receiving pre-WBI TLR9 agonist demonstrated improvement of survival after 10.4 Gy (p,0.03), 9.4 Gy (p,0.008) and 8.4 Gy (p,0.002) of WBI, compared to untreated or iMyd88-treated controls. Post-WBI TLR9 agonist mitigates up to 8.4 Gy WBI (p,0.01). Histological analysis and xylose absorption test demonstrated significant structural and functional restitution of the intestine in WBI+TLR9 agonist cohorts. Although, AIR reduced tumor growth, all animals died within 12 days from RIGS. TLR9 agonist improved the survival of mice beyond 28 days post-AIR (p,0.008) with significant reduction of tumor growth (p,0.0001). Conclusions: TLR9 agonist treatment could serve both as a prophylactic or mitigating agent against acute radiation syndrome and also as an adjuvant therapy to increase the therapeutic ratio of abdominal Radiation Therapy for Gastro Intestinal malignancies.
Funding: This work was supported in part by the National Institutes of Health (NIH) grants RC2 AI087612 and U19AI091175 to CG. No other external funding was
received for this study. Part of the findings of the manuscript has been presented in the 51st Annual Meeting of ASTRO in Chicago and was selected for the Poster
Recognition Award in Biology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Radiation-induced gastrointestinal syndrome (RIGS) remains
one of the major limitations for delivering tumoricidal doses of
abdominal radiation therapy (RT). It could also limit survival of
victims in a mass casualty setting from nuclear accidents or
terrorism. While supportive care with antibiotics, hydration and
bone marrow transplantation can rescue radiation-induced bone
marrow syndrome, currently there are no approved therapy for
protecting or mitigating RIGS. Manifestations of RIGS are
influenced by radiation dose with a short prodromal syndrome
of nausea and vomiting upon exposure to whole body irradiation
of 1.5 Gy. With higher doses ($6 Gy), the prodromal syndrome is
more marked, followed by a subacute syndrome of diarrhea and
gastrointestinal bleeding in 25 days post-exposure. As doses reach
over 810 Gy, the full-blown syndrome of RIGS with diarrhea,
dehydration, sepsis and intestinal bleeding ensues with eventual
mortality. In the clinic, higher doses from fractionated RT or large
single fractions during sterotactic body radiation therapy (SBRT)
can cause diarrhea and gastrointestinal bleeding, ulcer or fistula
from breakdown of irradiated intestinal mucosa. The efficacy of
cancer treatment by radiation and chemotherapeutic drugs is often
limited by severe side effects that primarily affect the
hematopoietic system and the epithelium of the gastrointestinal tract.
Progress in understanding differences in the mechanisms involved
in the responses of normal and tumor cells to genotoxic stress has
led to the development of new rational approaches to selective
protection of normal cells, such as suppression of apoptosis by
pharmacological inhibition of p53 or activation of NF-k B.
Another promising approach presented in this issue by Johnson
et al. is based on the idea of using pharmacological inhibitors of
cyclin-dependent kinases (CDKs) to convert normal cells into a
radioresistant state by inducing reversible cell cycle arrest at the
The pathophysiological mechanisms of RIGS is complex and
involves loss of clonogenic crypt cells with eventual depopulation
of the intestinal villi, defective regeneration of the irradiated
intestinal stem cells and a systemic inflammatory response
syndrome (SIRS) from a host of cytokines and growth factors
released in the serum, following exposure to radiation and gut
microbes [1,2]. Survival from RIGS depends on the rate of the
crypt depopulation and the efficiency and number of the residual
clonogens, capable of regenerating crypt-villus units. Therefore,
growth factors that can promote proliferation of intestinal crypt
cells, such as, keratinocyte growth factor and interleukin-11 have
been shown to be radioprotective for RIGS [3,4,5]. Similarly,
pretreatment with growth factors that inhibit cell cycle in regenerating
crypts, such as, transforming growth factor-b1 (TGFb1) and
transforming growth factor-b3 (TGFb3), promoted radioresistance
in these cells. Besides the intestinal crypts where the putative
intestinal stem cell (ISC) resides, the stroma or the ISC niche has
also been postulated to be a target in RIGS. Thus, endothelial cells
residing in the ISC niche, is particularly vulnerable to radiation.
Growth factors, such as, basic fibroblast growth factor (bFGF) that
prevented radiation-induced endothelial [6,7,8,9,10] cell death,
also conferred radioprotection from RIGS.
Besides the endothelial cell the intestinal subepithelial immune
effector cells, such as, gd-T cells and macrophages interact with
the host enteric microbiota and modulate the intestinal
regenerative response following radiation exposure . Cellular signaling
via the Toll-like receptor (TLR) and other pathogen-associated
molecular pattern (PAMP) recognition molecules plays a critical
role in the dynamic interactions between the hosts enteric
microbiota and innate immune system [12,13]. Intestinal
macrophages function as mobile cellular transceivers that recognize
invading bacteria entering through the disrupted intestinal mucosa
via the TLR receptors and transmit regenerative and repair signals
to the neighboring intestinal epithelial progenitors in the ISC
niche by forming immune synapses with the ISC . Recently, a
peptide derived from Salmonella Flagellin, which is a ligand for
TLR5, was found to be radioprotective against RIGS in murine
and primate models, possibly via NFkB activation . TLR9 is
present in the basolateral surface of crypt epithelial cells and could
potentially sense bacterial invasion via TLR ligand binding.
Earlier reports demonstrated that activation of TLR9 pathway by
bacterial CpG-rich oligonucleotides could be beneficial in
protecting against chemical models of intestinal injury .
TLR stimulation could also contribute to generation of
antiinflammatory signal and dampen intestinal sepsis . We,
therefore, hypothesized that systemic administration of TLR9
ligands could activate TLR signaling in the intestine and confer
radioprotection and/or mitigation from RIGS. In this study we
have used a novel TLR9 agonist containing synthetic
immunomodulatory CpR (R = 29-deoxy-7-dezaguanosine) dinucleotide
and 39-39-attached novel structures that has been shown to induce
potent TLR9-mediated immune responses [18,19,20,21]. The
presence of 39-39-attached structure provides higher metabolic
stability and also optimal 59-end presentation required for TLR9
recognition [18,22,23,24]. These novel TLR9 agonists have been
shown to induce potent Th1-type immune responses and a broad
spectrum of antitumor activity in a number of tumor models
[18,25,26,27,28]. A human selective TLR9 agonist, referred to as
IMO-2055, is in phase II clinical trials for cancer . Here we
demonstrate that activation of the TLR9 pathway by a novel
TLR9 agonist promoted intestinal crypt cell survival by inhibiting
radiation-induced apoptosis and stimulated regeneration of the
46 weeks old C57/Bl6 and BALB/c male mice were
purchased from Jackson Laboratory (Portland, Main), and housed
in the barrier facility at the Albert Einstein College of Medicine
(Bronx, NY). Mice were maintained ad libitum and all studies were
performed under the guidelines and protocols of the Institutional
Animal Care and Use Committee of the Albert Einstein College of
Medicine. The animal use protocol for this study was reviewed
and approved by the Institutional Animal Care and Use
Committee (IACUC) of Albert Einstein College of Medicine
(IACUC approval# 20080703).
Cell lines and maintenance
CT26 a mice colon carcinoma cell line (ATCC, Manassas, VA),
IEC6 rat intestinal epithelial cell line (ATCC) and J774A.1 a
mouse macrophage cell line (ATCC) were cultured according to
ATCC recommended protocol. In brief CT26 was cultured in
RPMI-1640 growth Medium and J774A.1, IEC6 were cultured in
Dulbeccos Modified Eagles Medium in presence of 10% fetal
bovine serum. Cells were maintained in 37uC in presence of 5%
TLR9 agonist Treatment
TLR9 agonist was synthesized, purified and characterized at
Idera Pharmaceuticals as described previously [18,30]. The purity
of the final product was 95% with less than 0.1 EU/ml of
endotoxin as determined by the Limulus assay (Bio-Whittaker
Lonza Walkersville Inc., Walkersville, MD). TLR9 agonist was
subcutaneously administered to C57Bl6 mouse (n = 15), 1 hour
prior to or after whole body irradiation (WBI) at a dose of 1.0 mg/
kg of body weight/mice. Control mice received subcutaneous
injection of phosphate buffered saline (PBS) (Manassas, VA). In
separate cohorts of mice (n = 15), we also inhibited the
downstream signaling of TLR9 by administering a
PBS-solubilized, MyD88 homodimerization inhibitory peptide,
DRQIKIWFQNRRMKWKKRDVLPGT (Imegenex, San Diego, CA).
Treatment with inhibitory peptide (0.5 mg/kg of body wt/
alternate day) was for 21 days prior to irradiation and continued
up to survival endpoint.
Animals were anesthetized by intraperitoneal injection of 100 ml
of ketamine and xylazine (7:1 mg/ml/mouse) and were exposed to
whole-body irradiation (WBI) 137Cs c-rays at a dose rate of
236 cGy/min (8.410.4 Gy). Abdominal irradiation (AIR) was
performed using a 320 KvP, Phillips MGC-40 Orthovoltage
irradiator at a 50 cm SSD with a 2 mm copper filter and a dose
rate of 72 cGy/min. AIR was administered to the mice after
shielding the thorax, head and neck and extremities and protecting
a significant portion of the bone marrow, thus inducing
The intestine of each animal was dissected, washed in PBS to
remove intestinal contents and the jejunum was fixed in 10%
neutral buffered formalin prior to paraffin embedding. Tissue was
routinely processed and cut into 5 mm sections for hematoxylin
and eosin and immunohistochemical staining. All hemotoxylin
and eosin (Fisher Scientific, Pittsburgh, PA) staining was
performed at the Histology and Comparative Pathology Facility in
the Albert Einstein Cancer Center.
In vivo BrdU labeling and Proliferation rate assay
To visualize proliferation, each mouse was injected
intraperitoneally with BrdU (120 mg/1 kg body weight, Sigma-Aldrich, St.
Louis, MO), 24 hrs prior to sacrifice and tissue harvest. BrdU
staining was performed on the sections. Briefly, 5 mm paraffin
sections were prepared from the mid-jejunum. After paraffin
removal and rehydration, tissue sections were incubated overnight
at room temperature with a biotinylated monoclonal BrdU
antibody (Zymed, San Francisco, CA). Streptavidin-peroxidase
was used as a signal generator and diaminobenzidine (DAB) was
used as a chromogen to stain BrdU-incorporated nuclei dark
brown. Sections were counterstained with hematoxylin,
dehydrated and mounted. Sections from mice not injected with BrdU were
used as the negative control.
Crypts were identified histologically according to the criteria
established by Potten C. S. . Pictures of crypts were taken at
high magnification Zeiss AxioHOME microscope and crypt
epithelial cells (paneth and non-paneth) in mouse intestinal
sections were examined using imageJ software and classified as
BrdU positive if they had brown-stained nuclei from DAB staining
or BRDU negative if they had blue stained nuclei. The
Proliferation rate was calculated as a percentage of BRDU
positive cells over the total number of cells in each crypt. A total of
30 crypts were examined per animal for all histological
parameters. A regenerative crypt was confirmed by
immunohistochemical detection of BrdU incorporation into five or more
epithelial cells within each crypt, scored in a minimum of four
cross-sections per mouse (n = 3/group).
Determination of Crypt depth
Crypt depth was scored by taking pictures of crypts in H&E
stained slides at 106 using a calibrated Zeiss AxioHOME
microscope. The images were analyzed using imageJ software to
measure the height in pixels from the bottom of the crypt to the
cryptvillus junction. This measurement in pixels was converted to length in
mM by with the following conversion factor: 1.46 pixels/mM.
Detection of apoptosis in tissues in situ
To assess the number of apoptotic cells, in situ detection of cells
with DNA strand breaks was performed in 4%-formalin-fixed,
paraffin-embedded intestinal section by the TUNEL (TdT
mediated digoxigenin labeled dUTPnick and labeling) technique
at Albert Einstein College of Medicine Dept. of pathology core
facility. In brief, paraffin embedded section were de-paraffinized,
rehydrated and stained using an ApopTag kit (Intregen Co,
Norcross, Georgia). The Apoptosis rate in crypt cells was quantified
by counting the percent of apoptotic cells in each crypt. Scoring was
restricted to good longitudinal sections of the crypt in which the base
of the crypt was aligned with all the other crypt bases, and evidence
showed the crypt lumen to be present [32,33].
Immunohistochemistry to detect Macrophage
Pericryptal macrophages were stained by Alexa
Fluor488conjugated anti-mouse, F480 antibody (1:50; Biolegend, San
Diego, CA). Images were captured using a Zeiss SP2 confocal
microscope at 406 optical zoom and the macrophages were
counted by using the VolocitySoft Version 5.0 (Improvision,
Waltham, MA) in 10 fields per mice in various cohorts (n = 3).
Nucleus was stained with DAPI and pseudo colored with red.
Xylose Absorption Assay
Functional regeneration of the irradiated intestines were
determined by measuring intestinal absorption by a xylose uptake
assay . To quantify intestinal absorption as a physiological
indicator of mucosal barrier integrity in different treatment group
(n = 5/group) a xylose uptake assay was performed, at various time
points (1, 3.5, 7 and 10 days) after irradiation. A 5% w/v solution
of D-xylose (100 ml/mouse) in deionized water was administered
orally by feeding tube. Animals were sacrificed after 2 hrs of
Dxylose administration and blood samples were collected using
heparinized blood collection tubes (BD Biosciences, San Jose, CA).
Fifty mL of serum sample was added to 5 ml of phloroglucinol
(1,3,5-trihydroxybenzene, Sigma Chemical Co., St. Louis, MO)
color reagent (0.5 g of phloroglucinol, 100 ml glacial acetic acid
and 10 ml of conc. HCL) heated to 100uC in a water bath for
4 min to allow optimum color development. After equilibration to
room temperature, sample absorption was determined with the aid
of a spectrophotometer set at a wavelength of 554 nm.
In vitro clonogenic assay
Immediately after irradiation IEC6 cells were plated in 9.6 cm2
culture plate. The number of cells seeded depended on the
radiation dose, so as to obtain a constant number of clones (50
100). The irradiated cells were allowed to grow until the surviving
cells produced macroscopic colonies that could be readily counted,
usually after 1014 days following irradiation. Only colo- nies
containing 50 or more cells were counted and the surviving
Figure 1. TLR9 agonist treatment protected C57Bl/6 mice from radiation-induced mortality. Kaplan-Meier survival analysis of C57Bl/6
mice treated with TLR9 agonist prior to WBI (A) 8.4 Gy; (B) 9.4 Gy and (C) 10.4 Gy. A significant survival was observed in TLR9 agonist-treated group,
exposed to WBI of 10.4 Gy (p,0.03), 9.4 Gy (p,0.008), 8.4 Gy (p,0.002) (log rank test) compared to WBI alone or WBI+TLR9 agonist+iMyd88 cohorts.
Treatment with TLR9 agonist after radiation exposure (WBI+Post-TLR9 agonist) had mitigation effect only at 8.4 Gy WBI. Administration of inhibitory
peptide against Myd88 (iMyd88) completely inhibited radioprotective effect of TLR9 agonist.
fraction was calculated as follows: Surviving fraction = colonies
counted/cell seeded6PE/100, where PE is the plating efficiency.
Kaplan-Meier Survival Curve Analysis
The effect of irradiation and concomitant TLR9 agonist on
mice survival/mortality was analyzed by Kaplan-Meier as a
function of radiation dose using SigmaPlot and Graphpad
Prism4.0 software for Apple PC.
TLR9 agonist improves survival after WBI
RIGS presents with diarrhea, weight loss and death within 514
days in mice exposed to higher WBI doses . We, therefore,
administered escalating doses of single fraction WBI (8.4, 9.4 and
10.4 Gy) to C57Bl/6J mice to induce RIGS. Irradiated animals
showed signs and symptoms of RIGS, including, diarrhea, black
stools and weight loss. Mortality increased in control mice that
received WBI and supportive treatment (antibiotics and PBS) with
100%, 80% and 0% of animals surviving 14 days, after 8.4, 9.4
and 10.4 Gy irradiation, respectively. Since TLR signaling
modulates intestinal regeneration, we examined whether a
TLR9-specific agonist, can protect against radiation lethality.
Mice receiving TLR9 agonist, prior to WBI, had well-formed
stools, maintained body weight (21.960.4 g in TLR9
agonist+WBI vs 15.1860.4 g in WBI cohort, p,0.008) with a
significant improvement in survival after 8.4 Gy (p,0.002),
9.4 Gy (p,0.008) and 10.4 Gy (p,0.03, log-rank test; Fig. 1)
WBI. Mitigation effect of TLR9 agonist was only seen at 8.4 Gy
WBI (p,0.01). In order to confirm the radioprotective role of
TLR9 signaling, we inhibited a universal adapter protein, Myd88,
which participates in TLR signaling by activating downstream
NFkB. Intravenous injection of an inhibitory peptide that disrupts
Myd88 homodimerization resulted in inhibition of radioprotection
with eighty percent of mice dead within 14 days of TLR9
agonist+Myd88 inhibitior+10.4 Gy WBI, suggesting involvement
of TLR9 signaling in mediating radioprotection (Fig. 1).
TLR9 agonist reduces intestinal crypt cell apoptosis in
Since ionizing radiation induces apoptosis of intestinal crypt
epithelial cells, we performed TUNEL assay to examine apoptosis
of crypt epithelial cells, 1 and 3.5 day after WBI. There was a
significant (p,0.001) decrease in the number of apoptotic nuclei in
the jejunal crypts of TLR9 agonist+WBI-treated animals
(11.6%62.11) as compared with the irradiated controls
(30.10%65.74) (n = 3), suggesting that TLR9 agonist might
increase the radio-resistance of the intestinal crypt compartment
by inhibiting radiation-induced apoptosis (Figure 2 A&D).
TLR9 agonist augments intestinal regeneration following
The characteristic tissue response to irradiation in intestine
consists of apoptosis of ISCs and enterocytes, resulting in crypt
depletion within day 1, followed by regeneration of surviving crypt
clonogens, resulting in crypt microcolony formation at 34 days
and eventual villi denudation by 7 days. We, therefore, examined
the histopathology of irradiated intestines at days 1, 3.5 and 7 with
hematoxylin-eosin staining and BrdU immunohistochemistry
(n = 3). Compared to irradiated controls, the percentage of the
BrdU-positive, crypt epithelial cells undergoing DNA synthesis
was significantly (p,0.002) enhanced after TLR9 agonist
treatment at 3.5 days post-WBI (Figs. 2A&C). This resulted in
an increase in crypt depth and size (p,0.001), suggesting that
TLR9 agonist treatment restitutes the crypt villous structures after
radiation exposure (Figs. 2A&B).
TLR9 agonist promotes recovery of intestinal absorption
in irradiated mice
To evaluate the functional regeneration and absorptive
capacity of the intestine, animals were fed xylose solution at
various time points post-WBI. Since xylose is not metabolized in
the body, serum xylose levels are a good indicator of the intestinal
absorptive capacity. Two hours after a test feeding-dose, serum
xylose levels were 13064 mg/ml in non-irradiated mice. As
expected, there was a progressive reduction in xylose absorption
in irradiated mice with serum xylose level of 5863.2 mg/ml in 7
days post-WBI. In contrast, mice treated with TLR9 agonist
before WBI exhibited a significant recovery of xylose absorption
106.464.03 mg/ml (p,0.0007) at this time point. Xylose
absorption continued to improve in the TLR9 agonist+WBI
cohort up to 10 days post-WBI (Fig. 3), indicating quick
restitution of the intestinal villi.
TLR9 agonist enhances the recruitment of intestinal
Intestinal pericryptal macrophages function as mobile cellular
transceivers that coordinate inputs from luminal microbes and
injured epithelium and transmit regenerative signals to
neighboring stem cell and stem cell niche . Moreover, activation of
these macrophages was dependent on TLR activating signals
coming from luminal microbiota. Immunohistological staining of
jejunal cross-section showed a decrease in intestinal macrophage
level in irradiated mice compared to non-irradiated control
(p,0.009). However, TLR9 agonist treatment prior to WBI
increased (p,0.006 at 1 day post WBI and p,0.001 at 3.5 day
post WBI) F480+ve cells in lamina propria and pericryptal region
suggesting a possible recruitment of intestinal pericryptal
macrophages in WBI+TLR9 agonist-treated mice (Fig. 4).
Transplantation of TLR9 agonist-treated macrophage
improves survival of mice exposed to WBI
We hypothesized that TLR9 agonist-mediated activation of
TLR9 in intestinal pericryptal macrophages results in secretion of
paracrine or trophic growth factors that induce crypt regeneration.
To evaluate whether TLR9 agonist-activated macrophages were
important to combat radiation damage, a macrophage cell line,
J774A.1, was first stimulated in vitro with TLR9 agonist incubation
for 624 hrs. Thereafter, TLR9 agonist-activated J774A.1 cells
were transplanted by intravenous injection in syngeneic BALB/c
(26106 cells/mice) mice, 1 hr after exposure to WBI (8.410.4 Gy).
Transplantation of TLR9 agonist-activated J774A.1 cells rescued
mice exposed upto 9.4 Gy WBI with significant improvement in
survival compared to irradiated mice receiving either no cell
transplantation (p,0.002) or J774A.1 cells that were not activated
by TLR9 agonist (p,0.005) (n = 10) (Fig. 5A). This suggests that
potential paracrine growth factors that are secreted by TLR9
agonist-treated J774A.1 cells might induce intestinal regeneration
after irradiation. We confirmed this by incubating rat intestinal
epithelial cells (IEC6) with culture supernatants from TLR9
agonisttreated J774A.1 cells following 26 Gy of irradiation in an in-vitro
clonogenic assay and demonstrated an increase in clonogenic
survival of irradiated IEC6 cells (survival fraction at 6 Gy
0.00960.004 vs 0.0860.01 p,0.0004) (Fig. 5B).
TLR9 agonist increases the therapeutic ratio of
Having demonstrated intestinal radioprotective effect of TLR9
agonist, we examined whether this effect by TLR9 agonist confers
radioprotection to abdominal tumors. A murine colorectal, CT26
tumor cell line was injected subcutaneously under the abdominal
Figure 4. TLR9 agonist treatment increase the number of macrophages in pericryptal and lamina propria of intestine of mice
treated with WBI. 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 (406) were magnified 2.36 (inset).
wall of BALB/c mice. Mice with palpable, subcutaneous tumors
were treated with TLR9 agonist, followed by whole abdominal
irradiation (AIR) of 14 Gy . AIR reduced the tumor growth
but invariably produced 100% mortality of animals with a median
survival time of 861.6 days. Compared to AIR alone, mice
receiving TLR9 agonist 1 hr prior to AIR showed significant
improvement in survival (median survival time 1962 days vs
861.6 days). In AIR+TLR9 agonist treated group 50% of mice
survived more than 30 days (Fig. 6B) and showed significant tumor
growth retardation compared to untreated non-irradiated control
(p,0.0001) (n = 10) (Fig. 6A). Comparison of the tumor volume
upto day 12 post AIR (as 100% mice were died in AIR cohort by
that day) showed significant difference between AIR and
AIR+TLR9 agonist group (20364.7 mm3 vs 8563.8 mm3
p,0.003). Therefore these results demonstrates that
administration of novel TLR9 agonist prior to irradiation may increase the
radiosensitivity of the colorectal tumors while providing
radioprotection to the normal tissues, including small intestine, thus
demonstrating an increase in the therapeutic ratio of abdominal
Figure 5. TLR9 agonist-activated macrophage mitigates RIGS. A. 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. Culture supernatant of TLR9 agonist-activated
J774A.1 induces clonogenic survival of rat intestinal IEC6 following irradiation. 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
(26 Gy) compared to irradiated control.
Figure 6. TLR9 agonist treatment improves the therapeutic ratio of whole abdominal RT for murine colorectal tumors. 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
The gastrointestinal system is an early response organ to
radiation. Rapid turnover of intestinal epithelial cells makes the
intestinal mucosa especially vulnerable to high radiation exposure
during RT or in nuclear accidents or terrorism. Thus,
maintenance of intestinal homeostasis is very critical to combat against
radiation-induced gastrointestinal injury. We have shown earlier
that administration of an intestinal stem cell growth factor
augment intestinal crypt cell proliferation in irradiated mice and
improve survival by ameliorating RIGS . In this report, we
demonstrate that activation of TLR signaling by a synthetic ligand
of TLR9 recruits pericryptal macrophages and provides growth
stimuli to promote intestinal regeneration and inhibit apoptosis of
crypt cells following radiation. TLR9 agonist treatment resulted in
an increase in intestinal crypt depth and proliferative index and a
decrease in crypt epithelial apoptosis with maintenance of the villi
length following WBI. There was a marked and progressive
restoration of the normal absorptive function of the intestine, as
measured by xylose absorption test. TLR9 agonist-treated mice
continued to survive beyond 46 weeks indicating that
pretreatment with TLR9 agonist could ameliorate radiation-induced bone
marrow syndrome in addition to RIGS. Besides radioprotection,
TLR9 agonist treatment could mitigate RIGS to doses up to
8.4 Gy WBI. Finally, in a model of murine colorectal CT26
tumor, pretreatment with TLR9 agonist protected the mice from
lethal RIGS, while improving the tumor suppression after whole
abdominal RT. A possible cause of tumor radiosensitization could
be potential immunomodulation, as demonstrated by Milas et al in
murine colon, breast and lung cancer models .
Selective protection of normal tissue from radiation toxicity has
also been achieved by modulating cell cycle arrest with different
pharmacological agents . Pharmacologic inhibitors of
cyclindependent kinases (CDKs) induces reversible cell cycle arrest at
the G1/S transition to convert normal cells into a transiently
growth arrested, radioresistant state, where as tumor cells showed
irreversible quiescence in response to these agents leading to
induction of senscence or apoptosis. In separate studies, it has been
shown that nutlin3, a P53 stabilizing agent, can be used in
combination with mitotic inhibitors to induce reversible cell cycle
arrest and thereby, cytoprotetion in normal cells, but not in P53
mutant tumor cells . A differential stress resistance has also
been observed between tumor cells and normal cells where
shortterm starvation or fasting induced chemoresistance in normal cells,
while tumor cells remained sensitive to chemotoxicity . It was
postulated that mutated oncogenes prevented the switch to a
protective stress response in tumor cells upon starvation for 24
48 hrs, while protecting normal cells across various model
organisms, in humans, mice and yeast [40,41]. Cancer cells are
more resistant to apoptosis from acquiring mutations in genes,
such as, p53 or inducing anti-apoptotic genes . Upon
genotoxic stress, normal cells with intact p53 either exhibit growth
arrest or die by apoptosis, while tumor cells are indifferent to
inhibition of apoptosis and are more prone to senescence .
Thus the anti-apoptotic role of TLR9 agonist could protect the
crypt cells from radiation-induced apoptotic death but does not
affect the colon cancer cells, which undergoes cell death or
senescence after radiation exposure. TLR9-mediated
immunomodulation might further enhance the therapeutic ratio of
Our study suggests that commensal intestinal bacteria could
play a significant role in maintaining intestinal homeostasis after
radiation exposure. Activation of TLRs by commensal micobiota
has been shown to augment mucosal restitution and modulate
inflammatory response in sepsis in response to various forms of
injury including radiation injury [44,45,46], necrotizing
enterocolitis or experimental colitis [16,47]. Pericryptal macrophages
act as an important member of the ISC niche and crosstalks with
intestinal subepithelial myofibroblast and ISC is essential to
maintain intestinal epithelial homeostasis. These pericryptal
macrophages recognize pathogen-associated molecular pattern
(PAMP) signals from invading microbial organisms after
radiation-induced mucosal injury. Various receptors, such as, TLR are
critical for recognizing PAMP and activating macrophages to
deliver regenerative signals to neighboring crypt ISCs . Our
results demonstrate that the universal TLR downstream adapter
molecule Myd88 is involved in RIGS protection because
inhibition of MyD88 homodimerization with an inhibitory
peptide suppressed the radioprotective effects of TLR9 agonist.
While, activation of NF-kB with the degradation of IkBa has
been implicated as downstream pathway to TLR-Myd88
signaling in injury models, such as, dextran sulfate sodium
(DSS)-induced experimental colitis , alternate pathways that
involve inhibition of NF-kB have also been implicated in
Growth factors, such as, R-spondin1, KGF, TGFb and bFGF
are known to protect the intestine from radiation or other
cytotoxic injury by increasing the crypt cell proliferation and
reducing apoptosis [5,7,35,36]. Clonogenic survival of a rat
intestinal epithelial cell, IEC6 was increased after incubation with
culture supernatants of TLR9 agonist-activated macrophage cell
line. Moreover, transplantation of TLR9 agonist-stimulated
macrophages induced radio-mitigation against 9.4 Gy WBI,
suggesting that TLR9 agonist-activated macrophages could secrete
growth factors that might mediate the radioprotective effects of
TLR9 agonist and TLR activation.
The vertebrate innate immune system has evolved to recognize
highly conserved repeated molecular patterns in cell walls and
nucleic acid structures of microbial pathogens using PAMP
receptors, such as, TLRs. Of the various TLRs, TLR9 recognizes
cytosine-phosphate-guanine (CpG) dinucleotides present in
bacterial DNA and synthetic oligomers. Kandimalla et al synthesized
designer TLR9 agonist containing two 59 ends and synthetic
purine bases, cytosine-phosphate-29-deoxy-7-deazaguanosine
dinucleotide motifs and demonstrated that these designer TLR9
agonists are potent ligands of TLR9 and are immunomodulatory
in mouse, monkeys and humans . The present report
demonstrates that these TLR9 agonists could serve both as a
prophylactic or mitigating agent against acute radiation syndrome
and also as an adjuvant therapy to increase the therapeutic ratio in
patients undergoing abdominal RT for GI malignancies.
Conceived and designed the experiments: SS CG EK SA. Performed the
experiments: SS PB LL DY EK. Analyzed the data: SS PB CG.
Contributed reagents/materials/analysis tools: DY EK SA CG. Wrote
the paper: SS PB AA EK CG.
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