Identification of granulocyte colony-stimulating factor and interleukin-6 as candidate biomarkers of CBLB502 efficacy as a medical radiation countermeasure.

Given an ever-increasing risk of nuclear and radiological emergencies, there is a critical need for development of medical radiation countermeasures (MRCs) that are safe, easily administered, and effective in preventing and/or mitigating the potentially lethal tissue damage caused by acute high-dose radiation exposure. Because the efficacy of MRCs for this indication cannot be ethically tested in humans, development of such drugs is guided by the Food and Drug Administration's Animal Efficacy Rule. According to this rule, human efficacious doses can be projected from experimentally established animal efficacious doses based on the equivalence of the drug's effects on efficacy biomarkers in the respective species. Therefore, identification of efficacy biomarkers is critically important for drug development under the Animal Efficacy Rule. CBLB502 is a truncated derivative of the Salmonella flagellin protein that acts by triggering Toll-like receptor 5 (TLR5) signaling and is currently under development as a MRC. Here, we report identification of two cytokines, granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6), as candidate biomarkers of CBLB502's radioprotective/mitigative efficacy. Induction of both G-CSF and IL-6 by CBLB502 1) is strictly TLR5-dependent, 2) occurs in a CBLB502 dose-dependent manner within its efficacious dose range in both nonirradiated and irradiated mammals, including nonhuman primates, and 3) is critically important for the ability of CBLB502 to rescue irradiated animals from death. After evaluation of CBLB502 effects on G-CSF and IL-6 levels in humans, these biomarkers will be useful for accurate prediction of human efficacious CBLB502 doses, a key step in the development of this prospective radiation countermeasure.

Resistance of bone marrow stroma to genotoxic preconditioning is determined by p53.

Transplantation of bone marrow (BM) is made possible by the differential sensitivity of its stromal and hematopoietic components to preconditioning by radiation and/or chemotherapeutic drugs. These genotoxic treatments eliminate host hematopoietic precursors by inducing p53-mediated apoptosis but keep the stromal niche sufficiently intact for the engraftment of donor hematopoietic cells. We found that p53-null mice cannot be rescued by BM transplantation (BMT) from even the lowest lethal dose of total body irradiation (TBI). We compared structural changes in BM stroma of mice differing in their p53 status to understand why donor BM failed to engraft in the irradiated p53-null mice. Irradiation did not affect the general structural integrity of BM stroma and induced massive expression of alpha-smooth muscle actin in mesenchymal cells followed by increased adiposity in p53 wild-type mice. In contrast, none of these events were found in p53-null mice, whose BM stroma underwent global structural damage following TBI. Similar differences in response to radiation were observed in in vitro-grown bone-adherent mesenchymal cells (BAMC): p53-null cells underwent mitotic catastrophe while p53 wild-type cells stayed arrested but viable. Supplementation with intact BAMC of either genotype enabled donor BM engraftment and significantly extended longevity of irradiated p53-null mice. Thus, successful preconditioning depends on the p53-mediated protection of cells critical for the functionality of BM stroma. Overall, this study reveals a dual positive role of p53 in BMT: it drives apoptotic death of hematopoietic cells and protects BM stromal cells essential for its functionality.

Accelerated thymic atrophy as a result of elevated homeostatic expression of the genes encoded by the TNF/lymphotoxin cytokine locus.

TNF, lymphotoxin (LT)-alpha, LT-beta and LIGHT are members of a larger superfamily of TNF-related cytokines that can cross-utilize several receptors. Although LIGHT has been implicated in thymic development and function, the role of TNF and LT remains incompletely defined. To address this, we created a model of modest homeostatic overexpression of TNF/LT cytokines using the genomic human TNF/LT locus as a low copy number Tg. Strikingly, expression of Tg TNF/LT gene products led to profound early thymic atrophy characterized by decreased numbers of thymocytes and cortical thymic epithelial cells, partial block of thymocyte proliferation at double negative (DN) 1 stage, increased apoptosis of DN2 thymocytes and severe decline of T-cell numbers in the periphery. Results of backcrossing to TNFR1-, LTbetaR- or TNF/LT-deficient backgrounds and of reciprocal bone marrow transfers implicated both LT-alpha/LT-beta to LTbetaR and TNF/LT-alpha to TNFR1 signaling in accelerated thymus degeneration. We hypothesize that chronic infections can promote thymic atrophy by upregulating LT and TNF production.

Immune response to MMTV infection.

Mouse mammary tumor virus (MMTV) has developed a strategy of exploitation of the immune response. It infects dendritic cells and B cells and requires this infection to establish an efficient chronic infection. This allows transmission of infection to the mammary gland, production in milk and infection of the next generation via lactation. The elaborate strategy developed by MMTV utilizes several key elements of the normal immune response. Starting with the infection and activation of dendritic cells and B cells leading to the expression of a viral superantigen followed by professional superantigen-mediated priming of naive polyclonal T cells by dendritic cells and induction of superantigen-mediated T cell B cell collaboration results in long-lasting germinal center formation and production of long-lived B cells that can later carry the virus to the mammary gland epithelium. Later in life it can induce transformation of mammary gland epithelium by integrating close to proto-oncogenes leading to their overexpression. Genes encoding proteins of the Wnt-pathway are preferential targets. This review will put these effects in the context of a normal immune response and summarize important facts on MMTV biology.

Redundancy in tumor necrosis factor (TNF) and lymphotoxin (LT) signaling in vivo: mice with inactivation of the entire TNF/LT locus versus single-knockout mice.

Homologous genes and gene products often have redundant physiological functions. Members of the tumor necrosis factor (TNF) family of cytokines can signal activation, proliferation, differentiation, costimulation, inhibition, or cell death, depending on the type and status of the target cell. TNF, lymphotoxin alpha (LTalpha), and LTbeta form a subfamily of a larger family of TNF-related ligands with their genes being linked within a compact 12-kb cluster inside the major histocompatibility complex locus. Singly TNF-, LTalpha-, and LTbeta-deficient mice share several phenotypic features, suggesting that TNF/LT signaling pathways may regulate overlapping sets of target genes. In order to directly address the issue of redundancy of TNF/LT signaling, we used the Cre-loxP recombination system to create mice with a deletion of the entire TNF/LT locus. Mice with a triple LTbeta/TNF/LTalpha deficiency essentially manifest a combination of LT and TNF single-knockout phenotypes, except for microarchitecture of the spleen, where the disorder of lymphoid cell positioning and functional T- and B-cell compartmentalization is severer than that found in TNF or LT single-knockout mice. Thus, our data support the notion that TNF and LT have largely nonredundant functions in vivo.

The Toll-Like Receptor 5 Agonist Entolimod Mitigates Lethal Acute Radiation Syndrome in Non-Human Primates.

There are currently no approved medical radiation countermeasures (MRC) to reduce the lethality of high-dose total body ionizing irradiation expected in nuclear emergencies. An ideal MRC would be effective even when administered well after radiation exposure and would counteract the effects of irradiation on the hematopoietic system and gastrointestinal tract that contribute to its lethality. Entolimod is a Toll-like receptor 5 agonist with demonstrated radioprotective/mitigative activity in rodents and radioprotective activity in non-human primates. Here, we report data from several exploratory studies conducted in lethally irradiated non-human primates (rhesus macaques) treated with a single intramuscular injection of entolimod (in the absence of intensive individualized supportive care) administered in a mitigative regimen, 1-48 hours after irradiation. Following exposure to LD50-70/40 of radiation, injection of efficacious doses of entolimod administered as late as 25 hours thereafter reduced the risk of mortality 2-3-fold, providing a statistically significant (P<0.01) absolute survival advantage of 40-60% compared to vehicle treatment. Similar magnitude of survival improvement was also achieved with drug delivered 48 hours after irradiation. Improved survival was accompanied by predominantly significant (P<0.05) effects of entolimod administration on accelerated morphological recovery of hematopoietic and immune system organs, decreased severity and duration of thrombocytopenia, anemia and neutropenia, and increased clonogenic potential of the bone marrow compared to control irradiated animals. Entolimod treatment also led to reduced apoptosis and accelerated crypt regeneration in the gastrointestinal tract. Together, these data indicate that entolimod is a highly promising potential life-saving treatment for victims of radiation disasters.

Prevention and mitigation of acute radiation syndrome in mice by synthetic lipopeptide agonists of Toll-like receptor 2 (TLR2).

Bacterial lipoproteins (BLP) induce innate immune responses in mammals by activating heterodimeric receptor complexes containing Toll-like receptor 2 (TLR2). TLR2 signaling results in nuclear factor-kappaB (NF-κB)-dependent upregulation of anti-apoptotic factors, anti-oxidants and cytokines, all of which have been implicated in radiation protection. Here we demonstrate that synthetic lipopeptides (sLP) that mimic the structure of naturally occurring mycoplasmal BLP significantly increase mouse survival following lethal total body irradiation (TBI) when administered between 48 hours before and 24 hours after irradiation. The TBI dose ranges against which sLP are effective indicate that sLP primarily impact the hematopoietic (HP) component of acute radiation syndrome. Indeed, sLP treatment accelerated recovery of bone marrow (BM) and spleen cellularity and ameliorated thrombocytopenia of irradiated mice. sLP did not improve survival of irradiated TLR2-knockout mice, confirming that sLP-mediated radioprotection requires TLR2. However, sLP was radioprotective in chimeric mice containing TLR2-null BM on a wild type background, indicating that radioprotection of the HP system by sLP is, at least in part, indirect and initiated in non-BM cells. sLP injection resulted in strong transient induction of multiple cytokines with known roles in hematopoiesis, including granulocyte colony-stimulating factor (G-CSF), keratinocyte chemoattractant (KC) and interleukin-6 (IL-6). sLP-induced cytokines, particularly G-CSF, are likely mediators of the radioprotective/mitigative activity of sLP. This study illustrates the strong potential of LP-based TLR2 agonists for anti-radiation prophylaxis and therapy in defense and medical scenarios.

Distinct and nonredundant in vivo functions of TNF produced by t cells and macrophages/neutrophils: protective and deleterious effects.

Tumor necrosis factor (TNF, TNFalpha) is implicated in various pathophysiological processes and can be either protective, as in host defense, or deleterious, as in autoimmunity or toxic shock. To uncover the in vivo functions of TNF produced by different cell types, we generated mice with TNF ablation targeted to various leukocyte subsets. Systemic TNF in response to lipopolysaccharide was produced mainly by macrophages and neutrophils. This source of TNF was indispensable for resistance to an intracellular pathogen, Listeria, whereas T-cell-derived TNF was important for protection against high bacterial load. Additionally, both T-cell-derived TNF and macrophage-derived TNF had critical and nonredundant functions in the promotion of autoimmune hepatitis. Our data suggest that T-cell-specific TNF ablation may provide a therapeutic advantage over systemic blockade.

Novel tumor necrosis factor-knockout mice that lack Peyer's patches.

We generated a novel tumor necrosis factor (TNF) null mutation using Cre-loxP technology. Mice homozygous for this mutation differ from their "conventional" counterparts; in particular, they completely lack Peyer's patches (PP) but retain all lymph nodes. Our analysis of these novel TNF-knockout mice supports the previously disputed notion of the involvement of TNF-TNFR1 signaling in PP organogenesis. Availability of TNF-knockout strains both with and without PP enables more definitive studies concerning the roles of TNF and PP in various immune functions and disease conditions. Here, we report that systemic ablation of TNF, but not the presence of PP per se, is critical for protection against intestinal Listeria infection in mice.

Transcriptional Regulation of the Gene, Encoding p40 Subunit of IL-12 and IL-23, in Murine Macrophages.

Interleukin-12 (IL-12) is an immunomodulatory cytokine with broad spectrum of activities, central of which are stimulation of NK cells, and promoting differentiation of T helper cells towards Th1 phenotype. IL-12 consists of two unrelated subunits, p35 and p40. Recently discovered cytokine, IL-23, which has both unique properties and those overlapping with IL-12, also contains p40 associated with another subunit, p19. Both IL-12 and IL-23 transmit the signal through receptors associated with JAK-STAT pathways. One of the critical components in the control of both IL-12 and IL-23 regulation is the transcriptional control of p40 gene which is induced in macrophages and dendritic cells in response to bacterial endotoxin, IFN-gamma and other stimuli. In the present study we report on the structure of the transcriptional unit of the murine p40 gene, its promoter and inducible expression of p40 in murine macrophages. Our findings are consistent with multi-level regulation of p40 expression.

SMUCKLER/TIM4 is a distinct member of TIM family expressed by stromal cells of secondary lymphoid tissues and associated with lymphotoxin signaling.

Lymphotoxin-alpha (LTalpha) was originally linked to delayed-type hypersensitivity and its production was later attributed to Th1, but not Th2 cells. Studies employing knockout mice demonstrated that LT signaling is essential for the development and functional compartmentalization of lymphoid tissues. Here, using gene expression profiling, we identified a novel gene termed SMUCKLER (spleen, mucin-containing, knockout of lymphotoxin), that is selectively down-regulated in spleens of LTalpha- or LTbeta-deficient mice. The encoded transmembrane protein contains immunoglobulin V and mucin domains and is identical to TIM4, a predicted member of recently identified TIM family (T cell immunoglobulin- and mucin-domain-containing molecule). Unlike TIM1 and TIM3, which were implicated in T cell-mediated functions, SMUCKLER lacks tyrosine phosphorylation motif in its intracellular domain and is not expressed by bone marrow-derived cells. In situ hybridization of spleen sections demonstrated SMUCKLER expression by stromal cells predominantly in the marginal zone and to a lesser extent throughout the white pulp. Similarly to other TIM genes, SMUCKLER maps to a locus associated with predisposition to asthma both in mice and in humans (11.b1 and 5q33, respectively) and shows coding sequence variations between BALB/c and DBA mice. Therefore, SMUCKLER/TIM4 may be considered as a candidate disease-predisposition gene for asthma.

Dissecting the role of lymphotoxin in lymphoid organs by conditional targeting.

Mice with inactivation of lymphotoxin beta receptor (LTbetaR) system have profound defects in the development and maintenance of peripheral lymphoid organs. As surface LT is expressed by lymphocytes, natural killer cells, and lymphoid tissue-initiating cells as well as by some other cell types, we dissected cell type-specific LT contribution into the complex LT-deficient phenotype by conditional gene targeting. B-LTbeta knockout (KO) mice displayed an intermediate phenotype in spleen as compared with mice with complete LTbeta deficiency. In contrast, T-LTbeta KO mice displayed normal structure of the spleen. However, inactivation of LTbeta in both T and B cells resulted in additional defects in the structure of the marginal zone and in the development of follicular dendritic cells in spleen. Structure of lymph nodes (LN) and Peyer's patches (PP) was normal in both B-LTbeta KO and T- and B-LTbeta KO mice, except that PPs were of reduced size. When compared across the panel of lymphocyte-specific LT KOs, the defects in antibody responses to T-cell-dependent antigens correlated with the severity of defects in spleen structure. Expression of CCL21 and CCL19 chemokines was not affected in spleen, LN and PP of B-LTbeta KO and T- and B-LTbeta KO mice, while CXCL13 was slightly reduced only in spleen. Collectively, our data suggest the following: (i). requirements for LT signaling to support architecture of spleen, LN and PP are different; (ii). LT complex expressed by B cells plays a major role in the maintenance of spleen structure, while surface LT expressed by T cells provides a complementary but distinct signal; and (iii). in a non-transgenic model, expression of lymphoid tissue chemokines is only minimally dependent on the expression of surface LT complex on B and T lymphocytes.

Distinct contributions of TNF and LT cytokines to the development of dendritic cells in vitro and their recruitment in vivo.

TNF/LTalpha/LTbeta (tumor necrosis factor/lymphotoxin-alpha/lymphotoxin-beta) triple knockout (KO) mice show a significant reduction of dendritic cell (DC) number in the spleen, presumably due to defective recruitment and/or production. To distinguish between these possibilities, DCs were generated from bone marrow (BM) cultures prepared from wild-type (wt) and mutant mice in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The yield of CD11c(+) major histocompatibility complex (MHC) class II(+) DCs generated from TNF/LTalpha/LTbeta(-/-) BM culture was significantly reduced compared with wt BM culture. In order to further dissect the individual pathways responsible for defective DC properties observed in TNF/LTalpha/LTbeta(-/-) mice, the panel of TNF/LT ligand and receptor single KO mice were used. The production of DCs from BM culture was significantly reduced in TNF(-/-) and TNF receptor (TNFR) p55(-/-) mice, but normal in LTalpha(-/-), LTbeta(-/-), LTbetaR(-/-) mice. Recombinant TNF (rTNF) exogenously added to TNF/LTalpha/LTbeta(-/-) BM cultures could reverse this defect, and blocking antibodies showed partial effect on BM cultures of wt mice. Conversely, numbers of mature DCs in spleen were significantly decreased in LTalpha(-/-), LTbeta(-/-), LTbetaR(-/-) mice, but not in TNF(-/-) and TNFRp55(-/-) mice. These results reveal 2 distinct contributions of TNF/LT cytokines. First, TNF acting through TNF receptor is involved in the development/maturation of DCs in BM progenitor cultures, but this function appears to be redundant in vivo. Second, the microenvironment in peripheral lymphoid organs associated with LTalpha/LTbeta-LTbetaR signaling and chemokine production is critical for recruitment efficiency of DCs, and this pathway is indispensable.