Central role of liver in anticancer and radioprotectiveactivities of Toll-like receptor 5 agonistLyudmila G. Burdelyaa,b, Craig M. Bracketta, Bojidar Kojouharova, Ilya I. Gitlina, Katerina I. Leonovaa,Anatoli S. Gleibermanb, Semra Aygun-Sunara, Jean Veitha, Christopher Johnsona, Gary J. Haderskib,Patricia Stanhope-Bakerb, Shyam Allamanenia, Joseph Skitzkia, Ming Zengc, Elena Martsenc, Alexander Medvedevc,Dmitry Scheblyakovd, Nataliya M. Artemichevad, Denis Y. Logunovd, Alexander L. Gintsburgd, Boris S. Naroditskyd,Sergei S. Makarovc, and Andrei V. Gudkova,b,1

aRoswell Park Cancer Institute, Buffalo, NY 14263; bCleveland BioLabs, Inc., Buffalo, NY 14203; cAttagene, Inc., Research Triangle Park, NC 27709;and dGamaleya Research Institute for Epidemiology and Microbiology, Moscow 123098, Russia

Edited by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved April 4, 2013 (received for review January 2, 2013)

Vertebrate Toll-like receptor 5 (TLR5) recognizes bacterial flagellinproteins and activates innate immune responses to motile bacte-ria. In addition, activation of TLR5 signaling can inhibit growth ofTLR5-expressing tumors and protect normal tissues from radiationand ischemia-reperfusion injuries. To understand the mechanismsbehind these phenomena at the organismal level, we assessednuclear factor kappa B (NF-κB) activation (indicative of TLR5 sig-naling) in tissues and cells of mice treated with CBLB502, a phar-macologically optimized flagellin derivative. This identified theliver and gastrointestinal tract as primary CBLB502 target organs.In particular, liver hepatocytes were the main cell type directly andspecifically responding to systemic administration of CBLB502 butnot to that of the TLR4 agonist LPS. To assess CBLB502 impact onother pathways, we created multireporter mice with hepatocytestransduced in vivowith reporters for 46 inducible transcription factorfamilies and found that along with NF-κB, CBLB502 strongly acti-vated STAT3-, phenobarbital-responsive enhancer module (PREM),and activator protein 1 (AP-1–) -driven pathways. Livers of CBLB502-treated mice displayed induction of numerous immunomodulatoryfactors andmassive recruitmentof various types of immunecells. Thisled to inhibition of growth of liver metastases of multiple tumorsregardless of their TLR5 status. The changed liver microenvironmentwas not, however, hepatotoxic, because CBLB502 induced resistanceto Fas-mediated apoptosis in normal liver cells. Temporary occlusionof liver blood circulation prevented CBLB502 from protecting hema-topoietic progenitors in lethally irradiated mice, indicating involve-ment of a factor secreted by responding liver cells. These resultsdefine the liver as the keymediator of TLR5-dependent effects in vivoand suggest clinical applications for TLR5 agonists as hepatoprotec-tive and antimetastatic agents.

breast cancer | colon cancer | neutrophils | natural killer cells | Salmonella

Toll-like receptors (TLRs) recognize and are activated by specificpatterns in molecules that are produced by a broad range of

microbial pathogens but are not present in host molecules. Acti-vation of TLRs by these pathogen-associated molecular patternsleads to induction of infection-fighting innate immune responses(1). Various TLR agonists have been considered for multiple clin-ical applications, including cancer immunotherapy (2–4), and one,the TLR7 agonist imiquimod, is approved for topical treatment ofbasal cell carcinoma (5).Although signaling pathways induced by different TLRs all

result in mobilization of an innate immune response and involveactivation of nuclear factor kappa B (NF-κB), the key regulatorof immunity (6, 7), TLR5 is a particularly attractive candidate fortherapeutic targeting for several reasons. First, bacterial flagellin,the natural ligand of TLR5, was found to have strong radiopro-tective effects in rodents and nonhuman primates (8). CBLB502is a rationally designed derivative of Salmonella flagellin that lacksthe highly immunogenic central globular domain and contains N-and C-terminal domains of the parental protein connected by

a flexible linker. It is substantially less immunogenic than full-length flagellin but retains its TLR5-dependent NF-κB–inducingactivity and radioprotective capability (8). CBLB502 (also calledEntolimod) is currently under development as a medical radia-tion countermeasure capable of both reducing damage to radio-sensitive hematopoietic (HP) and gastrointestinal (GI) tissuesand improving their regeneration. Moreover, CBLB502 pro-tected mice from dermatitis and mucositis associated with localfraction irradiation of head and neck area modeling radiationtreatment of patients with head and neck cancer (9). Second, theTLR5 agonist CBLB502 was shown to be effective as a tissueprotectant in mouse models of renal ischemia-reperfusion injury(10). Third, bacterial flagellin and flagellin-expressing bacteria(Salmonella) have shown antitumor effects in mouse models ofcolon and liver metastasis of pancreatic cancer (11, 12). Bacterialflagellin and its derivative, CBLB502, also demonstrated antitu-mor effects in several in vivo models (9, 13, 14). These effectsrequired TLR5 expression by the tumor cells and were pre-sumably mediated by innate immune cells recruited to the tumorfollowing activation of TLR5 and subsequent production of im-munomodulatory factors, such as cytokines. Fourth, TLR5 ago-nists are significantly less toxic than agonists of many other TLRs(e.g., the TLR4 agonist bacterial LPS). This is because “cytokinestorm”-inducing cytokines, such as tumor necrosis factor (TNF)

Significance

Toll-like receptor 5 (TLR5) is an innate immunity receptor thatspecifically recognizes and triggers immune response to bac-terial flagellins. In addition to resistance to Salmonella in-fection, TLR5 agonists protect mammals from radiation andhave anticancer effects, including suppression of tumor me-tastases. Using mouse models, we defined the liver as a majortarget for TLR5 agonists. Administration of pharmacologicallyoptimized flagellin derivative CBLB502 leads to rapid activationof prosurvival nuclear factor kappa B (NF-κB) and STAT3 path-ways in the liver and rescues mice from lethal doses of hepa-totoxic Fas-agonistic antibodies. Thus, TLR5 agonists can beconsidered for treatment and prevention of liver metastasis andhepatoprotective applications.

Author contributions: L.G.B., C.M.B., D.S., D.Y.L., A.L.G., B.S.N., S.S.M., and A.V.G. de-signed research; L.G.B., C.M.B., B.K., I.I.G., K.I.L., A.S.G., S.A.-S., J.V., G.J.H., S.A., M.Z., E.M.,A.M., D.S., N.M.A., and S.S.M. performed research; L.G.B., C.M.B., I.I.G., K.I.L., A.S.G., C.J., P.S.-B.,J.S., M.Z., E.M., A.M., D.S., N.M.A., D.Y.L., A.L.G., B.S.N., S.S.M., and A.V.G. analyzeddata; and L.G.B., C.M.B., P.S.-B., D.Y.L., S.S.M., and A.V.G. wrote the paper.

Conflict of interest statement: A.V.G. is a consultant and shareholder of ClevelandBioLabs, Inc., a biotech company that provided funding for this work.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: andrei.gudkov@roswellpark.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1222805110/-/DCSupplemental.

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and interleukin one beta (IL1-β), which are major determinantsof the toxicity associated with stimulation of other TLRs, are notincluded in the spectrum of cytokines induced following TLR5activation (15–17).To understand the reasons behind the specific physiological re-

sponse to and to facilitate rational exploration of potential clinicalapplications of TLR5 agonists, we sought to define the mechanismof action of CBLB502 in the context of the whole organism (usingthe mouse as a model system). Our focus was, foremost, on iden-tification of the tissues and cells that are direct primary respondersto CBLB502 and, subsequently, on analysis of themolecular effectsof CBLB502 in primary responders (e.g., induction of signalingpathways, production of bioactive factors). These goals weremet by

(i) analyzing tissue specificity of TLR5 response by using NF-κBreporter mice that enable detection of organ sites responding toCBLB502, (ii) an in vivo adaptation of the recently developedFACTORIAL assay (18) to monitor the response of multipletranscription factors to CBLB502 simultaneously, and (iii) a surgi-cal procedure for temporary occlusion of blood circulation throughthe liver to test the biological effects of factors secreted by liver cellsin response to CBLB502. This work defined the liver as the majorprimary target organ of CBLB502, leading to activation of severalprosurvival and immunoregulatory signaling pathways, as well asdramatic changes in the spectrum of secreted factors and immunecell content. CBLB502 treatment strongly suppressed growth oftumor cells in the liver regardless of their TLR5 status. These

Fig. 1. NF-κB activation and immune cell mobilization in response to CBLB502 and LPS. (A) Bioluminescent imaging of NF-κB–dependent luciferase expressionin BALB/c-Tg(IκBα-luc)Xen mice 2 h after s.c. injection of PBS or CBLB502 (0.2 mg/kg). (B) Luciferase activity in protein extracts of liver, small intestine (int.;ileum), large intestine (colon), kidneys, lungs, spleen, and heart tissue obtained from BALB/c-Tg(IκBα-luc)Xen mice 2 h after s.c. injection of PBS, CBLB502 (0.2mg/kg), or LPS (1 mg/kg) (n = 3 per group). Bars represent average ± SD. (C) Immunohistochemical detection of NF-κB p65 (green) nuclear translocation in liversections obtained from NIH Swiss mice 20, 40, or 60 min after s.c. injection of PBS [untreated (u/t)], CBLB502 (1 μg per mouse), or LPS (10 μg per mouse).Sections were costained with anti-cytokeratin 8 (ker8; red; epithelial cell marker) and DAPI (blue). Arrowheads indicate Kupffer and endothelial cells de-termined by morphological criteria. (Magnification: 40×.) (D) Immunohistochemistry with anti-p65 (green) and anti-cytokeratin 8 (red) in cultures of primaryhepatocytes from NIH Swiss mice or humans treated with PBS (u/t), CBLB502 (100 ng/mL), or LPS (1 μg/mL) for 1 h. (Magnification: 40×.) (E) FACS analysis ofNK-cell, neutrophil, and macrophage (Kupffer cell) populations in the liver and BM and T cells in the liver of mice treated with PBS or CBLB502 (1 μg, s.c.) forthe indicated amounts of time. The absolute number of cells per organ (whole liver and BM from two hind tibias and two hind femurs) is indicated. Error barsrepresent mean ± SEM (n = 6 mice per group). An asterisk indicates that the difference from intact mice is significant (P < 0.05).

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results, together with the finding that blood circulation through theliver is indispensable for CBLB502-mediated radioprotection ofthe HP system, revealed that the biological effects of TLR5 ago-nists are mediated, at least in part, by factors secreted by responsiveliver hepatocytes. In addition to providing mechanistic insightsregarding the radioprotective and anticancer activities of TLR5agonists, this study defined hepatoprotection as a specific area oftheir prospective clinical application.

ResultsLiver Is the Major Organ That Responds to the TLR5 Agonist CBLB502.Stimulation of TLR5 is known to activate the major immuno-regulatory transcription factor NF-κB, making NF-κB activationan accurate reflection of TLR5 agonist activity (1, 19). There-fore, to identify tissues that are primary responders to CBLB502

in vivo, we used noninvasive imaging to detect NF-κB–dependentluciferase expression in live BALB/c-Tg(IκBα-luc)Xen reportermice following injection of PBS vehicle or CBLB502. Intenseluciferase expression, indicative of strong NF-κB activation, wasobserved specifically in the livers of CBLB502-treated mice at 2 hpostinjection (Fig. 1A).To quantitate NF-κB activity in different tissues, ex vivo lu-

ciferase activity assays were performed using tissue lysates fromBALB/c-Tg(IκBα-luc)Xen reporter mice treated with PBS orCBLB502 for different amounts of time. For comparison, theTLR4 agonist LPS, which also activates NF-κB, was used atabout 50% of its maximum tolerated dose. Again, the livershowed the strongest NF-κB response to CBLB502. Substantialreporter induction was also observed in the large intestines ofCBLB502-treated mice (Fig. 1B). In contrast, LPS activated NF-κB specifically in the lungs, spleen, and kidney. In all responsive

Fig. 2. Activation of gene regulatory pathways in mouse liver in vivo by CBLB502 and LPS. (A) Schematic illustration of the in vivo FACTORIAL assay used toprofile changes in the activities of transcription factors in the mouse liver following in vivo CBLB502 or LPS treatment. Two weeks after library transfection,mice were injected with CBLB502 (5 μg per mouse), LPS (10 μg per mouse), or PBS, and RTU activities in total liver RNA samples were isolated 1 h later andanalyzed for the RTU activity as previously described (18). Description of transcription factors listed in the table can be found in www.attagene.com/cis-1-list.pdf. (B) The radial graph shows fold-induction values of mean activities of individual RTUs in CBLB502 or LPS treated mice vs. vehicle-treated mice (n = 8 miceper group). A fold induction value of 1× indicates that the pathway was not affected by the applied treatment. (C) mRNA levels for several apoptosis-relatedfactors and cytokines implicated in TLR5 signaling (and GAPDH as a control) were analyzed by RT-PCR using total RNA from livers of untreated mice (“time 0”)or mice treated with CBLB502 (1 μg per mouse) for 30 min or 120 min; bcl2a1b, B cell leukemia/lymphoma 2 related protein A1b; bcl2a1d, B cell leukemia/lymphoma 2 related protein A1d; jun, jun proto-oncogene; cxcl1, chemokine (C-X-C motif) ligand 1; il6, interleukin 6; gapdh, glyceraldehyde-3-phosphatedehydrogenase.

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tissues, the kinetics of reporter activation were similar for bothTLR agonists, peaking ∼2 h postinjection, declining by 6 h, andreturning to near baseline by 24 h (data for representative organsare shown in Fig. S1).To define specific CBLB502-responsive cell types within the

liver, we used immunohistochemistry to detect nuclear trans-location of the p65 subunit of NF-κB as an indicator of NF-κBactivation. At 20 min after CBLB502 injection (the earliest testedtime point), NF-κB translocation was observed in essentially allhepatocytes but not in other liver cell types, including Kupffer andendothelial cells (Fig. 1C). The response was opposite in LPS-treated mice: Early translocation of NF-κB was observed inKupffer and endothelial cells but not in hepatocytes. Later (by 1 hpostinjection), all liver cells in both TLR5 and TLR4 agonist-treated mice displayed nuclear NF-κB accumulation, presumablyreflecting combined primary effects of TLR agonists on cellsexpressing their cognate receptors and secondary effects mediatedby factors released by primary responding cells. NF-κB trans-location was observed in pure cultures of primary hepatocytes frommice and humans (Fig. 1D) following treatment with CBLB502 butnot LPS, regardless of the duration of treatment. These resultsindicate that liver hepatocytes of rodents and primates expressfunctional TLR5 but not TLR4 and that hepatocytes are majorspecific primary targets of TLR5 agonists.

Activation of Immunostimulatory and Tissue Protective Pathways inLivers of CBLB502-Treated Mice. To identify perturbations in sig-naling pathways in the mouse liver in response to TLR5 activa-tion, we used a multiplexed reporter system (the FACTORIAL)enabling a simultaneous assessment of multiple transcriptionfactor families within cells (18). As shown schematically in Fig.2A, FACTORIAL multireporter mice were generated via in vivotransfection of mouse hepatocytes [using hydrodynamic stress(20)] with a “library” of 46 constructs, each containing a reportertranscription unit (RTU) controlled by a minimal promoterpreceded by binding sites specific for a particular transcriptionfactor. FACTORIAL multireporter mice were treated withCBLB502, LPS, or PBS for 1 h, and the 46 RTU-encoded tran-scripts were quantified in total liver RNA. This revealed dramaticactivation of NF-κB (1,000-fold), STAT3 (200-fold), pheno-barbital-responsive enhancer module (PBREM) (200-fold),and activator protein 1 (AP-1) (50-fold) signaling in livers fromCBLB502-treated mice (Fig. 2B). In contrast, these pathwayswere either nonresponsive to LPS (for AP-1) or were at least 10-fold less responsive to LPS than to CBLB502. Rapid activation ofNF-κB, STAT3, and AP-1 in livers of CBLB502-treated mice wasconfirmed by global gene expression profiling (using Illuminamicroarray hybridization) at 30 min and 2 h after CBLB502 in-jection (several examples are shown in Table S1). Consistent withthe nature of activated transcription factors, this analysis showedstrong changes (predominantly increases) in the abundance ofmRNAs encoding multiple classes of bioactive factors, includingNF-κB–responsive immunomodulators (cytokines, chemokines,and their receptors) and antiapoptotic and antimicrobial factors.mRNA induction for representative genes was confirmed usingRT-PCR (Fig. 2C). Livers of CBLB502-treated mice also showedinduction of IL-6, an NF-κB–regulated cytokine known to induceSTAT3 signaling (21).These observations support our conclusion defining hep-

atocytes as primary responders to TLR5 agonists but not TLR4agonists and provide a rational basis for observed biological ac-tivities of TLR5 agonists (e.g., tissue protection).

TLR5 Agonist Treatment Leads to Mobilization of Immune Cells to theLiver. Because NF-κB is a major regulator of immune responsesand induces numerous immunostimulatory factors, we predictedthat NF-κB activation in the livers of CBLB502-treated micewould lead to recruitment of immune cells to the liver. This wasverified by FACS analysis of total liver cells and bone marrow

(BM), a known depot of immune cells, stained with specific Abcombinations (Fig. 1E). Five hours after CBLB502 treatment(the earliest tested time point), there was a 30% increase in theoverall cellularity of the liver (Fig. S2A). This was due, at least inpart, to rapid recruitment (by 5 h posttreatment or earlier) ofnatural killer (NK) cells and neutrophils to the liver (Fig. 1E).CBLB502-induced neutrophil accumulation was transient andcompletely resolved by 24 h. NK cells, however, remained ele-vated for at least 5 d (the duration of the experiment). Similarkinetics were observed for natural killer-T cells (Fig. S2B). Thenumber of macrophages in the liver was only slightly increased byCBLB502 treatment. In addition to effectors of innate immunity,we observed recruitment of adaptive immune cells; total con-ventional αβ+ T cells, including CD4+/CD8+ T cells, wererecruited to the liver within 5 h of CBLB502 injection andreturned to baseline by day 5 (Fig. 1E and Fig. S2B). In the BM,NK-cell, neutrophil, and macrophage populations all displayedpatterns of change following CBLB502 treatment that were op-posite of those observed in the liver, thus suggesting that the BMis a likely source of at least some of the immune cells recruited tothe liver.

CBLB502-Mediated Radioprotection of Hematopoietic Progenitor CellsIs Liver-Dependent. The observed CBLB502 responsiveness of theliver suggested that it might play a role in the radioprotectiveactivity of the compound by secreting CBLB502-induced bio-active factors. This possibility was supported by the failure ofCBLB502 to protect HP cells against radiation-induced death invitro (Fig. 3 A and B), despite its clear radioprotective effects onthe HP system in vivo (8) (Fig. 3B). For example, treatment ofprimary mouse bone marrow cells (BMCs) with CBLB502 in vitrodid not preserve granulocyte–macrophage colony formation po-tential (Fig. 3A). Moreover, although BM irradiated in the con-text of the whole animal showed CBLB502-dependent preservationof granulocyte–macrophage colony formation, BMCs that wereisolated from CBLB502-injected mice and then irradiated invitro did not (Fig. 3B).To test whether the liver response to CBLB502 is indeed

important for its radioprotective activity, we assessed CBLB502-mediated protection of HP progenitor cells in irradiated mice inwhich blood circulation through the liver was blocked during the30-min period of CBLB502 treatment and then restored. Theeffectiveness of this liver exclusion/reperfusion (LER) procedurewas confirmed by quantifying NF-κB–dependent luciferase levelsin liver tissue extracts from BALB/c-Tg(IκBα-luc)Xen reportermice 2 h after application of LER or sham-LER clamps andinjection of CBLB502 or PBS (Fig. S3). NIH Swiss mice werethen used to assess CBLB502-mediated HP radioprotectionunder conditions of LER and sham-LER. BMCs were obtainedfrom CBLB502- or PBS-injected mice immediately after irradi-ation and analyzed in granulocyte–macrophage colony formationassays. Under conditions of sham-LER, CBLB502 treatmentresulted in significantly greater postirradiation colony formationthan PBS treatment (Fig. 3C). However, under conditions ofliver exclusion, there was no difference between CBLB502 andPBS treatment; drastic radiation-induced depletion of HPprogenitors was observed in both cases (Fig. 3C). Injection ofCBLB502 under liver exclusion did not alter the colony-formingpotential of BMCs in nonirradiated mice, indicating that neitherthe drug nor the applied procedure had an adverse impact on theBM. The results obtained using LER strongly support our hy-pothesis that CBLB502-stimulated liver cells produce blood-borne factor(s) essential for protection of HP progenitor cellsfrom radiation damage.

TLR5 Agonist Treatment Suppresses Liver Metastasis. The liver isa frequent site for metastatic growth of multiple tumor types, andit is the primary site of colon cancer metastasis (22). Our ob-servation of CBLB502-induced recruitment of large numbers of

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immune cells to the liver suggested that the changed liver mi-croenvironment might affect the growth of liver metastases. Wetested this in several syngeneic mouse models of liver metastasis,including colon carcinoma CT26, lymphoma A20, and breastcarcinoma 4T1.

CT26 and A20 tumor cells were introduced into mice either bys.c. injection to mimic primary tumor formation or by intrasplenicinjection (followed by rapid spleen removal), which leads toformation of multiple liver nodules mimicking liver metastasesof the tumor. Formation of such experimental liver metastaseswas confirmed by bioluminescent imaging of live mice injectedwith luciferase-expressing CT26 cells and treated with PBS (Fig.4A). Neither CT26 nor A20 tumor cells express TLR5 mRNA orrespond to CBLB502 with NF-κB activation (Fig. S3). Consistentwith this result and with previous results showing that antitumoreffects of CBLB502 required expression of functional TLR5by the tumor, we found that CBLB502 treatment did not altergrowth of s.c. CT26-derived metastases (Fig. S4A). However,growth of CT26 metastases in the liver was suppressed byCBLB502 treatment on days 5 and 6 (Fig. 4 A and B, Top) or ondays 1, 3, and 5 (Fig. S4B). Both regimens of CBLB502 treat-ment significantly delayed tumor growth in livers and increasedthe proportion of animals that remained tumor-free for at least50 d. CBLB502 was even more effective in suppressing hepaticgrowth of A20 tumors. Mice treated with 1 μg of CBLB502 ondays 5 and 6 or on days 5–9 or with 5 μg of CBLB502 on days 5–9after intrasplenic A20 cell inoculation were >95% tumor-free onday 60, whereas only 40% of mice treated with vehicle weretumor-free (Fig. 4B, Bottom). These results illustrate a liver-specific antitumor effect of TLR5 agonists that does not requireTLR5 expression by the tumor cells.CBLB502 also suppressed spontaneous liver metastasis

of mouse breast adenocarcinoma 4T1 cells, which do expressfunctional TLR5 (Fig. S3). In this model, metastasis was eval-uated in a clinically relevant setup following surgical removal ofprimary tumors. The 4T1 cells were inoculated into mammaryfat pads of syngeneic BALB/c mice, and the mice were giventhree daily treatments of PBS or CBLB502 when primary tumorswere about 2 mm in diameter. Primary tumors were resectedwhen they reached 400 mm3 in volume, and the mice weretreated again with PBS or CBLB502 on days 1, 3, and 5 post-surgery. Twenty-one days after surgery, clonogenic assays wereperformed to quantify liver metastases as previously described(23). As shown in Fig. 4D, mice treated with CBLB502 afterprimary tumor resection (with either PBS or CBLB502 treat-ment before resection) had significantly fewer colony-forming4T1 cells in their livers compared with mice treated with PBSboth before and after resection (P < 0.05). Two out of four micetreated with CBLB502 before resection and with PBS after re-section also had significantly fewer 4T1 cells in their livers.Consistently, there was a significant increase in the proportionof animals that survived following surgical removal of primarytumors in CBLB502-treated groups (Fig. 4E), presumablyreflecting eradication of tumor cells infiltrating distal organsof tumor-bearing mice.

Metastasis Suppression by CBLB502 Is Mediated by Mobilization of NKCells to the Liver. To determine whether immune cell mobilizationplays a role in the observed suppression of liver metastasis inCBLB502-treated mice, we evaluated the morphology of mouselivers containing CT26 metastases. Luciferase-expressing CT26cells were delivered to mice by intrasplenic injection, and 20 dlater, when tumor growth in livers was detected by luminescentimaging, mice were given two daily s.c injections of PBS orCBLB502 (1 μg per mouse). At 5 h after the second injection,H&E-stained liver sections showed multiple metastases in liversof vehicle-treated animals (Fig. 4C, white arrows) characterizedby frequent mitoses within tumor cells (Fig. 4C, red arrows) andno visible inflammatory infiltrates. CBLB502-treated mice hadliver metastases of similar size and distribution (Fig. 4C, whitearrows) as vehicle-treated animals but also showed marked ac-cumulation of immune cells in the liver. Neutrophils and lym-phocytes were seen at the rim of growing metastases borderingthe liver parenchyma (Fig. 4C, green arrows). In some areas,

Fig. 3. CBLB502-mediated protection of BM granulocyte/macrophage (GM)progenitors from radiation damage. BM GM colony-forming units (GM-CFUs)were quantified as described in Materials and Methods. Bars in graphs rep-resent the average number of colonies in triplicate cultures ± SD. (A) GM-CFUsin BMCs isolated and treated in vitro with CBLB502 (100 ng/mL) or PBS vehicle,followed by 10-Gy irradiation 30 min later. (B) GM-CFUs in BMCs isolated im-mediately after 10-Gy total body irradiation (TBI) from the mice that wereinjected with either CBLB502 (1 μg per mouse) or PBS 30 min before irradia-tion. As indicated by an asterisk, BMCs in one regimen were isolated frommice30 min after CBLB502 injections and then irradiated in vitro with 10 Gy. (C)GM-CFUs in BMCs isolated from mice treated with the indicated combinationsof CBLB502 (1 μg per mouse) or PBS and 10-Gy TBI under conditions of LER(occlusion) or sham-treatment (surgery without occlusion).

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Fig. 4. Effect of CBLB502 treatment in experimental and spontaneous metastatic tumor models. (A) Bioluminescent imaging of CT26 tumors in livers ofBALB/c mice injected (s.c.) with PBS (untreated) or CBLB502 (0.04 mg/kg) on days 5 and 6 after intrasplenic tumor cell inoculation. Images shown arefrom day 14. (B) Proportion of mice without tumor growth in the liver was determined by bioluminescent imaging. (Top) Mice were injected as inA. (Middle) Following intrasplenic CT26 inoculation, mice were left untreated, injected with CBLB502, or treated with anti-asialo GM1 Ab in combi-nation with CBLB502 given on days 1, 3, and 5 (P value indicates the difference from intact and Ab-injected mice). (Bottom) A20 lymphoma cells weredelivered to BALB/c mice by intrasplenic injection. The combined results of three regimens of CBLB502 treatment [0.05 mg/kg of CBLB502 on days 5 and6 (n = 8) or on days 5–9 (n = 8) or 0.2 mg/kg of CBLB502 on days 5–9 (n = 9)] are shown. (C ) H&E-stained liver sections obtained from mice withestablished CT26 liver metastases were treated with vehicle (A and B) or CBLB502 (C and D). The areas boxed in A and C are shown in B and D.(Magnification: A and C, 10×; B and D, 40×.) Arrows indicate CT26 metastases (white), mitotic tumor cells (red), neutrophils and lymphocytes at theboundary between metastases and liver parenchyma (green), and extravasation of inflammatory cells from hepatic vessels into liver tissue (blue). (D)Quantification of spontaneous liver metastasis of 4T1 tumors in mice given two rounds of PBS or CBLB502 treatment. Clonogenic assays were performedby plating total liver cells in 6-thioguanine–containing medium. The mean number of 4T1 colonies per mouse as determined from triplicate wells isshown. P values were calculated for comparison with PBS-only treatment (paired t test). (E ) Mouse survival (%) was monitored during 75 d postsurgery.The difference between CBLB502-treated groups (n = 5 each) and PBS-injected (n = 4) mice was significant (log-rank test, P < 0.05).

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recruitment of inflammatory cells from hepatic vessels into theliver was evident (Fig. 4C, blue arrows).The importance of NK cells in particular for CBLB502-medi-

ated suppression of liver metastases was assessed inmice depletedof NK cells using antiasialo GM1 Abs (24), which specificallydepleted NK cells without affecting neutrophils or macrophages(Fig. S5). Mice were treated with anti-asialo GM1 Ab 1 d beforeand immediately before CBLB502 injection on days 1 and 5;CBLB502 injections (1 μg per mouse) were given on days 1, 3, and5 after intrasplenic injection of CT26 cells. Imaging (IVIS Im-aging System, 100 series; Xenogen Corp.) was used to monitordevelopment of CT26 metastases in the liver and showed that thepreviously observed antitumor effect of CBLB502 (Fig. 4B, Top)was completely abrogated by depletion of NK cells (Fig. 4B,Middle). This result is consistent with previous work demon-strating the importance of innate immune cells for TLR5-medi-ated antitumor effects (14).

CBLB502 Treatment Protects Mice from Fas-Mediated Hepatotoxicity.Cytotoxicity of activated immune cells, including those mobilizedto the liver by CBLB502 (e.g., NK cells, neutrophils), is oftenmediated by their expression of Fas ligand (25, 26). This, to-gether with the high sensitivity of liver cells to Fas-mediated

apoptosis (27, 28), raises the possibility that CBLB502-inducedinflammation in the liver might be hepatotoxic. However, on theother hand, TLR5 agonists are strong activators of NF-κB, whichhas powerful antiapoptotic effects, including protection againstFas-mediated apoptosis (29). To test the effect of CBLB502 onliver sensitivity to Fas-mediated apoptosis, we treated mice withanti-Fas agonistic Ab (anti-Fas Ab) at doses capable of inducinglethal hepatotoxicity associated with signs of apoptosis, livertissue necrosis, and hemorrhage (30, 31). Injection of NIH Swissmice with anti-Fas Ab resulted in 100% mortality within 1–2 d(Fig. 5A). In contrast, 100% of mice survived when they weregiven CBLB502 30 min or 2 h before anti-Fas Ab. CBLB502 hada lesser but still beneficial effect on survival when injected 10 minor 6 h before anti-Fas Ab. The protective effect of CBLB502 onnormal liver tissue in this model was confirmed using H&E stain-ing, TUNEL staining, and erythrocyte autofluorescence to detectnecrosis, apoptotic cells, and hemorrhagic lesions, respectively(Fig. 5C). In addition, Fas-induced elevation of serum alanineaminotransferase levels [an indicator of liver damage (32)] wassignificantly reduced by CBLB502 pretreatment (Fig. 5B).Fas-dependent apoptosis involves activation of caspase-8, which

leads to activation of effector caspase-3 and caspase-7 either di-rectly (in type I cells) or indirectly (in type II cells) via a chain of

Fig. 5. Protection against Fas-mediated hepatotoxicity by CBLB502. (A) Survival of NIH Swiss mice on day 20 after i.p. injection of 4 μg of anti-Fas Ab alone(PBS, n = 5) or in combination with CBLB502 (1 μg per mouse) injected s.c. 10 min (n = 5), 30 min (n = 16), 2 h (n = 10), or 6 h (n = 10) before anti-Fas injection.(B) Mice were treated with CBLB502 given 30 min before anti-Fas Ab. Serum alanine aminotransferase (ALT) levels were determined at 6 h after anti-Fasinjection (Top), caspase-8 (Middle) and caspase-3/7 (Bottom) activities in liver tissue protein lysates were determined at 5 h. Bars represent average ± SD (n =3–4 mice per group). For all three assays, the difference between mice given anti-Fas alone vs. anti-Fas + CBLB502 was statistically significant (P < 0.05). (C)Evaluation of histological sections of liver tissue prepared 5 h after injection of NIH Swiss mice with vehicle or with anti-Fas Ab alone or in combination withCBLB502. Tissue morphology was assessed by H&E staining; apoptotic cells were detected by TUNEL staining; and hemorrhage was visualized by erythrocyteautofluorescence (red), mouse IgG control staining (Cy5-conjugated anti-mouse IgG Ab, purple), and DAPI staining of nuclei (blue). (Magnification: 20×.) (D)Western blot analysis of full-length and cleaved Bid in liver tissue protein lysates from NIH Swiss mice left untreated (“vehicle”) or injected with anti-Fas Ab,CBLB502, or both. Lysates were prepared 2 h after anti-Fas injection.

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events involving BH3 interacting domain death agonist (Bid) ac-tivation, mitochondrial cytochrome c release, and caspase-9 acti-vation (33). CBLB502 pretreatment only slightly reduced caspase-8activation in livers of anti-Fas Ab–treated mice (Fig. 5B) and onlypartially suppressed cleavage of the endogenous caspase-8 target,Bid (Fig. 5D). In contrast, activation of caspase-3 and caspase-7 wasnearly eliminated (Fig. 5B; also observed in BALB/c and C57BL/6mice, Fig. S6). These results suggest that the liver cells affected byanti-Fas Ab are type II cells and that prevention of apoptosis byCBLB502 occurs predominantly downstream of caspase-8 andBid, presumably at the level of cytochrome c release, as pre-dictable by the observed induction of the B-cell lymphoma 2family proteins (Fig. 2C) and their upstream regulator, STAT3(Fig. 2B). These proteins are known to block production of re-active oxygen species (ROS) and suppress the mitochondrialapoptotic pathway (34). In addition, activation of STAT3 sig-naling in hepatocytes was previously shown to have an anti-apoptotic effect (35).Fas-mediated apoptosis has also been implicated in the path-

ogenicity of Salmonella infection in mice, which is predominantlylocalized in the liver (36–38). Consistent with this, treatment withCBLB502 (but not with LPS) improved survival of mice infectedwith a lethal dose of Salmonella typhimurium (Fig. S7). Thisprotective effect was likely due to a combination of suppression ofFas-mediated apoptosis in the liver by CBLB502 (as describedabove) and its ability to induce production of antimicrobialfactors (detected by microarray-based gene expression analysis;some examples are shown in Table S2; to be reported in detail ina separate paper).

DiscussionStimulation of TLR5 by its natural ligand, flagellin, or by the fla-gellin derivative CBLB502 causes NF-κB activation on the cellularlevel and multiple biological effects, including tissue protective andantitumor effects with strong clinical potential, on the organismallevel. Fundamental to understanding themechanism(s) of action ofTLR5 agonists is identification of tissues that are primary res-ponders to these agents. This report demonstrates that the strongestNF-κB activation in response to TLR5 agonist CBLB502 occurs inthe liver and GI tract. Although these observations possibly reflectthe tissue specificity of TLR5 expression, this assumption cannot bedirectly tested due to lack of a reliable immunohistochemical assayfor TLR5. Hepatocytes were shown to be responsible for the pri-mary CBLB502 response in the liver, and cells of the lamina propria

[presumably dendritic cells as reported by Uematsu et al. (39)] wereidentified as likely primary responders in intestinal tissue. Expres-sion and function of TLR5 in the liver make biological sense, giventhat the liver is the primary site of Salmonella residence duringinfections (36). Our finding that TLR5 and TLR4 are expressed intwo different nonoverlapping liver compartments, hepatocytes andKupffer cells, respectively, further differentiates TLR5 from TLR4,the latter of which is predominantly expressed in cells of the im-mune system (1). Operation of these two receptors in differenttissue microenvironments/epigenetic backgrounds provides a plau-sible explanation, along with differences in TLR5- and TLR4-activated signal transduction cascades, for the distinct biologicaloutcomes of TLR5 agonist (flagellin) and TLR4 agonist (LPS) ex-posure. This includes induction of different cytokines (1, 4), whichtranslates into cardinal differences in toxicity. Although LPSstrongly induces the highly toxic cytokines TNF and IL-1β, both ofwhich are capable of triggering a harmful cytokine storm (40–42),these cytokines are not induced by flagellin or CBLB502, whichpredominantly up-regulate granulocyte colony-stimulating factor(G-CSF), IL-6, IL-8, and IL-10 (8, 16, 17).FACTORIAL assay (18) combined with global gene expression

profiling identified multiple signaling pathways and downstreamgenes activated in hepatocytes of CBLB502-treated mice that areconsistent with observed biological effects of TLR5 stimulation. Inparticular, NF-κB–regulated immunomodulators, such as cyto-kines, are likely responsible for rapid andmassive infiltration of theliver with different types of immune cells following CBLB502treatment. In this way, CBLB502 has a negative effect on hepaticgrowth of tumor cells, regardless of their TLR5 expression status(Fig. 6). In contrast, in TLR5-negative environments outside of theliver, the tumor itself must express TLR5 to mobilize an antitu-mor immune response following TLR5 agonist treatment (9,14). The hypothetical scheme of CBLB502 tumor suppressiveactivity in the liver is shown in Fig. 6.The fact that the liver is a principal site for tumor metastasis

(22) and for residence of immature NK cells (43) adds to thesignificance of our finding that NK cells are critical for theactivity of CBLB502 against CT26 liver metastasis. NK cells areknown to participate in surveillance and elimination of tumorcells through their cytolytic activity and production of cytokinesthat affect other components of the innate and adaptive immunesystems (44). Cytolytic activity of NK cells against tumors cells istypically not antigen-dependent but may involve alternative re-ceptor-mediated recognition of “stress-induced” ligands com-monly expressed on tumor cells. The importance of neutrophils

Fig. 6. Schematic description of a plausible mechanism of the liver metastasis suppression effect of CBLB502.

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for the antitumor effects of TLR5 agonists noticed by others (13,14), together with our finding of neutrophils’ mobilization to theliver, suggests that cross-talk between neutrophils and NK cellsmay be involved. This notion is supported by previous workshowing the involvement of neutrophils in NK-cell function (45).In addition to regulating immunomodulatory factors, NF-κB

controls expression of numerous other factors, including anti-apoptotic proteins, scavengers of ROS, and growth factors (46).Such factors may play roles in CBLB502’s protection of (i) the HPand GI systems against radiation (8), (ii) the kidney againstischemia-reperfusion injury (10), and (iii) the liver againstFas-mediated apoptosis (this report). In these cases, additional“prosurvival” TLR5 agonist responses may include those con-trolled by the STAT3 (47, 48) and AP-1 pathways (49), whichwere previously shown to be responsible for liver resistance toFas-mediated apoptosis (35), as well as by the PBREM, whichregulates genes involved in the detoxifying metabolic capacity ofthe liver (50). The positive effect of TLR5 stimulation on liverresistance to severe stresses is illustrated by rescue of mice pre-treated with CBLB502 from otherwise lethal doses of Fas ago-nistic Ab. This result explains the lack of liver damage inCBLB502-treated animals regardless of massive mobilization tothis organ of inflammatory cells, the cytotoxic effect of which islargely mediated by their expression of Fas ligand (25, 26).Our studies testing the radioprotective effect of CBLB502

under conditions of temporary exclusion of the liver from bloodcirculation indicate that the liver is a critical source of solublefactors acting directly or indirectly (i.e., via modulation of cellulartrafficking) to protect the HP system against radiation damage(Fig. 3). The nature of these factors remains to be determined.However, likely candidates include the cytokines G-CSF and IL-6,which have been found elevated in the blood of CBLB502-treatedmice and monkeys (8) and are known stimulators of HP stemcells (G-CSF) and the thrombopoietic lineage of hematopoiesis(IL-6) (51–53).In addition to providing results consistent with other data and

predictable hypotheses, our in vivo FACTORIAL assay pointed topreviously unknown properties of CBLB502. For example, al-though not activated to the same level as the top four pathways(NF-κB, AP-1, STAT-3, and PBREM), the antioxidant responseelement [nuclear factor (erythroid-derived 2)-like 2 (NRF2)] RTUshowed a strong (20-fold) and rapid response to CBLB502 but notLPS (Fig. 2B). Based on the known downstream targets of thispathway, this may indicate early induction of antioxidant enzymes(e.g., superoxide dismutase, GST) in hepatocytes of CBLB502-treated animals (54). Secreted antioxidants produced in this waycould act as systemic ROS scavengers, and thus contribute to boththeHP radioprotective activity and liver tissue protective activity ofCBLB502. This possibility, as well as the significance of similarTLR5 agonist-specific induction of octamer-binding transcriptionfactor (Oct) pathway (Fig. 2B), will be explored in future studies.In summary, the key finding of this study, that hepatocytes are

a major and specific primary target of TLR5 agonists, providesinsight into the mechanism(s) of action of these agents, which havestrong potential as therapeutics for radiation protection and re-duction of cancer treatment side effects (8, 9); prevention of is-chemia-reperfusion injury (10); immunotherapy of TLR5-positivetumors (9, 13, 14); and, as revealed by this report, suppression ofliver metastases of different types of cancer regardless of theirTLR5 status and protection against hepatotoxic insults.

Materials and MethodsMice.NIH Swiss femalemice (National Cancer Institute) andBALB/c andC57BL/6female mice (Jackson Laboratory) aged 10–14 wk were used. Balb/C-Tg(IκBα-luc)Xen reporter mice were originally purchased from Xenogen Corp.and bred in our domestic colony. All animal experiments followed protocolsapproved by the Roswell Park Cancer Institute Institutional Animal Care andUse Committee.

Reagents. TLR5 agonistic agent CBLB502 was obtained from ClevelandBioLabs, Inc. (8, 55), and LPS from Escherichia coli 055:B5 was purchasedfrom Sigma. Purified hamster anti-mouse Fas Ab, clone Jo2, was purchasedfrom BD Biosciences. Purified anti-asialo GM1 Ab was purchased fromWako Chemicals.

Tumor Cells. Murine mammary carcinoma 4T1, B-cell lymphoma A20, andcolon undifferentiated carcinoma CT26 cells (American Type Culture Col-lection) were transduced with constitutive luciferase expression constructsand were cultured in RPMI plus 10% (vol/vol) FBS with standard supplementsand antibiotics.

Primary Hepatocytes. Primary human hepatocytes were purchased from BDBiosciences. Primary mouse hepatocytes were isolated from the liver of NIH-Swiss or BALB/c-Tg(IκBα-luc)Xen mice by collagenase digestion as describedpreviously (56). Hepatocytes were cultured in William’s modified E mediumsupplemented with 10% FBS, 1% penicillin/streptomycin, 2 mM glutamine,50 ng/mL EGF, 10 mM nicotinamide, 10−7 M dexamethasone, and 1× insulin-transferrin-selenite (57).

Imaging of Luciferase Expression in Live Mice. BALB/c-Tg(IκBα-luc)Xen micetreated with CBLB502 or PBS were injected i.p. with 15 mg/mL D-luciferin(Promega) in PBS (0.1-mL volume), anesthetized using 1–2% isoflurane, andimaged (within 5 min of D-luciferin injection) using an IVIS Imaging System andLiving Image software (Caliper LifeSciences). The same method was used todetect luciferase-expressing CT26 and A20 tumor cells in BALB/c mice.

Quantification of NF-κB–Driven Luciferase Expression in Protein Extracts. Tissuesamples of liver, lungs, kidney, spleen, heart, and intestine fromBALB/c-Tg(IκBα-luc)Xen mice treated with PBS, CBLB502, or LPS were homogenized in proteinlysis buffer (Promega) containing proteinase inhibitor mixture (Calbiochem).Luciferase activity was measured in 20 μL of lysate after adding 30 μL of lu-ciferin reagent (Bright-Glo Luciferase Assay System; Promega) and normalizedto the extract protein concentration measured using a BioRad DC protein assaykit II. Luciferase fold induction was calculated as the ratio between the aver-age normalized luciferase activity in organs of TLR ligand-treated mice andPBS-injected mice. To assess NF-κB activation in tumor cell lines, cells weretransiently transfected with the p5XIP10 κB reporter construct containing fivetandem copies of the NF-κB binding site from the interferon-inducible protein10 promoter upstream of the luciferase gene using Lipofectamine Plus (Invi-trogen Life Technologies). Luciferase activity was measured 5 h after the cellswere treated with CBLB502, LPS, or TNF using the Bright-Glo LuciferaseAssay System.

Immunohistochemical Staining of p65. Paraffin-embedded sections and pri-marymouse and human hepatocyteswere stainedwith a rabbit polyclonal Abagainst NF-κB p65 (catalog no. 7970; Abcam) and a rat monoclonal Ab againstcytokeratin 8 (Troma-1; Developmental Studies Hybridoma Bank, Universityof Iowa), followed by secondary fluorochrome-conjugated Abs [p65 (green)and cytokeratin-8 (red)] and DAPI (blue) DNA stain.

FACS Analysis of Immune Cell Populations. Single cell suspensions wereobtained frommouse livers by perfusion with PBS followed by digestion with0.05% collagenase type IV (Sigma–Aldrich) as described (58). Single cell sus-pensions of BMCs were obtained by flushing hind leg femurs and tibias withPBS. Following lysis of RBCs, cells were stained with a mixture of mAb againstCD45 (clone 30-F11), CD11b (clone M1/70), GR-1 (clone RB6-8C5), F4/80 (cloneBM8), CD63 (clone FA-11), CD3e (clone 145-2C11), bTCR (clone H57-597), andCD49b (clone DX5) (all from BioLegend); fixed in 2% (vol/vol) formalin; andanalyzed using a BD Biosciences Fortessa instrument andWinList 7.0 software(Verity Software House). Neutrophils were defined as CD45+, CD11b+, GR-1hi

(mean fluorescence intensity >103); macrophages were defined asCD45+CD11b+F4/80+; NK cells were defined as CD45+CD3e−CD49b+; and T cellswere defined as CD45+CD3e+αβTcR+.

FACTORIAL Analysis of Signaling Pathway Activity. A FACTORIAL library con-sisting of 46 different plasmids each containing a single unique pathway-specific reporter transcription unit (RTU) (Attagene, Inc., www.attagene.com)was transfected into livers of NIH Swiss mice by a “hydrodynamic” trans-fection trough the tail vein that predominantly targets the plasmids to liverhepatocytes (20). Mice were allowed to recover after transfection for 2 wkand then were injected (s.c.) with CBLB502 (5 μg per mouse), LPS (10 μg permouse), or PBS (eight mice per group). RTU activities were evaluated in totalliver RNA samples collected 1 h later using multiplex RT-PCR detection with

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RTU-specific primers (18). Changes in transcriptional factor activities werecalculated by normalizing the mean activities of RTUs in the livers of micetreated with CBLB502 or LPS to those in PBS-treated mice.

LER Procedure. LER was achieved by using a nontraumatic clamp to occludethe hepatoduodenal ligament containing the hepatic artery and portalvein (59). CBLB502 or PBS was injected; 30 min later, clamps were removedto restore liver blood circulation, abdomens were closed, and mice wereimmediately subjected to total body irradiation using a 137Cs Mark I-30

irradiator (J. L. Shepherd and Associates) with a dose rate of 2.2 Gy/min.Sham-LER was performed identically, with the exception that the hep-atoduodenal ligament was occluded.

ACKNOWLEDGMENTS. We thank the staff of the Department of LaboratoryAnimal Research (Roswell Park Cancer Institute) for their contribution to ourexperiments. This work was supported by National Institutes of HealthGrants R01AI080446 and RC2AI087616 and by Cleveland BioLabs, Inc.(A.V.G.), as well as by the Defense Threat Reduction Agency under ContractHDTRA1-11-C-0008 (to Cleveland BioLabs, Inc.).

1. Takeda K, Kaisho T, Akira S (2003) Toll-like receptors. Annu Rev Immunol 21:335–376.2. Curigliano G, et al. (2007) Vaccine immunotherapy in breast cancer treatment:

Promising, but still early. Expert Rev Anticancer Ther 7(9):1225–1241.3. Waterston AM, Cassidy J (2005) Adjuvant treatment strategies for early colon cancer.

Drugs 65(14):1935–1947.4. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R (2004)

Recognition of commensal microflora by toll-like receptors is required for intestinalhomeostasis. Cell 118(2):229–241.

5. Jiménez-Baranda S, Silva IP, Bhardwaj N (2012) Plasmacytoid dendritic cells lead thecharge against tumors. J Clin Invest 122(2):481–484.

6. Gilmore TD, Wolenski FS (2012) NF-κB: Where did it come from and why? ImmunolRev 246(1):14–35.

7. Hayden MS, Ghosh S (2011) NF-κB in immunobiology. Cell Res 21(2):223–244.8. Burdelya LG, et al. (2008) An agonist of toll-like receptor 5 has radioprotective activity

in mouse and primate models. Science 320(5873):226–230.9. Burdelya LG, et al. (2012) Toll-like receptor 5 agonist protects mice from dermatitis

and oral mucositis caused by local radiation: Implications for head-and-neck cancerradiotherapy. Int J Radiat Oncol Biol Phys 83(1):228–234.

10. Fukuzawa N, Petro M, Baldwin WM, 3rd, Gudkov AV, Fairchild RL (2011) A TLR5agonist inhibits acute renal ischemic failure. J Immunol 187(7):3831–3839.

11. Soto LJ, 3rd, et al. (2003) Attenuated Salmonella typhimurium prevents theestablishment of unresectable hepatic metastases and improves survival in a murinemodel. J Pediatr Surg 38(7):1075–1079.

12. Yam C, et al. (2010) Monotherapy with a tumor-targeting mutant of S. typhimuriuminhibits liver metastasis in a mouse model of pancreatic cancer. J Surg Res 164(2):248–255.

13. Cai Z, et al. (2011) Activation of Toll-like receptor 5 on breast cancer cells by flagellinsuppresses cell proliferation and tumor growth. Cancer Res 71(7):2466–2475.

14. Rhee SH, Im E, Pothoulakis C (2008) Toll-like receptor 5 engagement modulates tumordevelopment and growth in a mouse xenograft model of human colon cancer.Gastroenterology 135(2):518–528.

15. Akira S, Takeda K (2004) Functions of toll-like receptors: Lessons from KO mice. C RBiol 327(6):581–589.

16. Carvalho FA, Aitken JD, Gewirtz AT, Vijay-Kumar M (2011) TLR5 activation inducessecretory interleukin-1 receptor antagonist (sIL-1Ra) and reduces inflammasome-associated tissue damage. Mucosal Immunol 4(1):102–111.

17. Vijay-Kumar M, et al. (2008) Toll-like receptor 5-deficient mice have dysregulatedintestinal gene expression and nonspecific resistance to Salmonella-induced typhoid-like disease. Infect Immun 76(3):1276–1281.

18. Romanov S, et al. (2008) Homogeneous reporter system enables quantitativefunctional assessment of multiple transcription factors. Nat Methods 5(3):253–260.

19. Tallant T, et al. (2004) Flagellin acting via TLR5 is the major activator of key signalingpathways leading to NF-kappa B and proinflammatory gene program activation inintestinal epithelial cells. BMC Microbiol 4:33.

20. Zhang G, Budker V, Wolff JA (1999) High levels of foreign gene expression inhepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther 10(10):1735–1737.

21. Yu H, Pardoll D, Jove R (2009) STATs in cancer inflammation and immunity: A leadingrole for STAT3. Nat Rev Cancer 9(11):798–809.

22. Lise M, Mocellin S, Pilati P, Nitti D (2005) Colorectal liver metastasis: Towards theintegration of conventional and molecularly targeted therapeutic approaches. FrontBiosci 10:3042–3057.

23. Pulaski BA, Ostrand-Rosenberg S (1998) Reduction of established spontaneousmammary carcinoma metastases following immunotherapy with major histocompatibilitycomplex class II and B7.1 cell-based tumor vaccines. Cancer Res 58(7):1486–1493.

24. Kasai M, et al. (1981) In vivo effect of anti-asialo GM1 antibody on natural killeractivity. Nature 291(5813):334–335.

25. Berke G (1995) The CTL’s kiss of death. Cell 81(1):9–12.26. Smyth MJ, et al. (2005) Activation of NK cell cytotoxicity.Mol Immunol 42(4):501–510.27. Malhi H, Gores GJ, Lemasters JJ (2006) Apoptosis and necrosis in the liver: A tale of

two deaths? Hepatology 43(2 Suppl 1):S31–S44.28. Galle PR, et al. (1995) Involvement of the CD95 (APO-1/Fas) receptor and ligand in

liver damage. J Exp Med 182(5):1223–1230.29. Yin XM, Ding WX (2003) Death receptor activation-induced hepatocyte apoptosis and

liver injury. Curr Mol Med 3(6):491–508.30. Ogasawara J, et al. (1993) Lethal effect of the anti-Fas antibody in mice. Nature

364(6440):806–809.31. Nishimura M, Naito S (2005) Tissue-specific mRNA expression profiles of human toll-

like receptors and related genes. Biol Pharm Bull 28(5):886–892.

32. Ozer J, Ratner M, Shaw M, Bailey W, Schomaker S (2008) The current state of serumbiomarkers of hepatotoxicity. Toxicology 245(3):194–205.

33. Barnhart BC, Alappat EC, Peter ME (2003) The CD95 type I/type II model. SeminImmunol 15(3):185–193.

34. Lacronique V, et al. (1996) Bcl-2 protects from lethal hepatic apoptosis induced by ananti-Fas antibody in mice. Nat Med 2(1):80–86.

35. Taub R (2003) Hepatoprotection via the IL-6/Stat3 pathway. J Clin Invest 112(7):978–980.

36. Xu HR, Hsu HS (1992) Dissemination and proliferation of Salmonella typhimurium ingenetically resistant and susceptible mice. J Med Microbiol 36(6):377–381.

37. Shimizu H, et al. (2002) Toll-like receptor 2 contributes to liver injury by Salmonellainfection through Fas ligand expression on NKT cells in mice. Gastroenterology123(4):1265–1277.

38. Zbell AL, Maier SE, Maier RJ (2008) Salmonella enterica serovar Typhimurium NiFeuptake-type hydrogenases are differentially expressed in vivo. Infect Immun 76(10):4445–4454.

39. Uematsu S, et al. (2006) Detection of pathogenic intestinal bacteria by Toll-likereceptor 5 on intestinal CD11c+ lamina propria cells. Nat Immunol 7(8):868–874.

40. Beutler B, Milsark IW, Cerami AC (1985) Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science, 1985,229(4716):869-871. Classical article. J Immunol 181(1):7–9.

41. Dinarello CA, Orencole SF, Savage N (1989) Interleukin-1 induced T-lymphocyteproliferation and its relation to IL-1 receptors. Adv Exp Med Biol 254:45–53.

42. Le J, Vilcek J (1987) Tumor necrosis factor and interleukin 1: Cytokines with multipleoverlapping biological activities. Lab Invest 56(3):234–248.

43. Takeda K, et al. (2005) TRAIL identifies immature natural killer cells in newborn miceand adult mouse liver. Blood 105(5):2082–2089.

44. Terunuma H, Deng X, Dewan Z, Fujimoto S, Yamamoto N (2008) Potential role of NKcells in the induction of immune responses: Implications for NK cell-basedimmunotherapy for cancers and viral infections. Int Rev Immunol 27(3):93–110.

45. Jaeger BN, et al. (2012) Neutrophil depletion impairs natural killer cell maturation,function, and homeostasis. J Exp Med 209(3):565–580.

46. Karin M, Greten FR (2005) NF-kappaB: Linking inflammation and immunity to cancerdevelopment and progression. Nat Rev Immunol 5(10):749–759.

47. Takeda K, et al. (1998) Stat3 activation is responsible for IL-6-dependent T cellproliferation through preventing apoptosis: Generation and characterization ofT cell-specific Stat3-deficient mice. J Immunol 161(9):4652–4660.

48. Turkson J, Jove R (2000) STAT proteins: Novel molecular targets for cancer drugdiscovery. Oncogene 19(56):6613–6626.

49. Trierweiler C, Blum HE, Hasselblatt P (2012) The transcription factor c-Jun protectsagainst liver damage following activated β-Catenin signaling. PLoS ONE 7(7):e40638.

50. Gnerre C, et al. (2005) LXR deficiency and cholesterol feeding affect the expressionand phenobarbital-mediated induction of cytochromes P450 in mouse liver. J LipidRes 46(8):1633–1642.

51. Patchen ML, Fischer R, MacVittie TJ (1993) Effects of combined administration ofinterleukin-6 and granulocyte colony-stimulating factor on recovery from radiation-induced hemopoietic aplasia. Exp Hematol 21(2):338–344.

52. MacVittie TJ, et al. (1991) Cytokine therapy in canine and primate models ofradiation-induced marrow aplasia. Behring Inst Mitt (90):1–13.

53. Patchen ML, MacVittie TJ, Solberg BD, Souza LM (1990) Survival enhancement andhemopoietic regeneration following radiation exposure: Therapeutic approach usingglucan and granulocyte colony-stimulating factor. Exp Hematol 18(9):1042–1048.

54. Zhu H, Itoh K, YamamotoM, Zweier JL, Li Y (2005) Role of Nrf2 signaling in regulationof antioxidants and phase 2 enzymes in cardiac fibroblasts: Protection againstreactive oxygen and nitrogen species-induced cell injury. FEBS Lett 579(14):3029–3036.

55. Krivokrysenko VI, et al. (2012) Identification of granulocyte colony-stimulating factorand interleukin-6 as candidate biomarkers of CBLB502 efficacy as a medical radiationcountermeasure. J Pharmacol Exp Ther 343(2):497–508.

56. Gleiberman AS, Sharovskaya YuYu, Chailakhjan LM (1989) “Contact inhibition” ofalpha-fetoprotein synthesis and junctional communication in adult mouse hepatocyteculture. Exp Cell Res 184(1):228–234.

57. Fougère-Deschatrette C, et al. (2006) Plasticity of hepatic cell differentiation:Bipotential adult mouse liver clonal cell lines competent to differentiate in vitro andin vivo. Stem Cells 24(9):2098–2109.

58. Klein I, et al. (2007) Kupffer cell heterogeneity: Functional properties of bone marrowderived and sessile hepatic macrophages. Blood 110(12):4077–4085.

59. Abe Y, et al. (2009) Mouse model of liver ischemia and reperfusion injury: Methodfor studying reactive oxygen and nitrogen metabolites in vivo. Free Radic Biol Med46(1):1–7.

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