Immunosuppressive Mechanisms in Brucellosis in Light of Chronic Bacterial Diseases.

Brucellosis is considered one of the major zoonoses worldwide, constituting a critical livestock and human health concern with a huge socio-economic burden. Brucella genus, its etiologic agent, is composed of intracellular bacteria that have evolved a prodigious ability to elude and shape host immunity to establish chronic infection. Brucella's intracellular lifestyle and pathogen-associated molecular patterns, such as its specific lipopolysaccharide (LPS), are key factors for hiding and hampering recognition by the immune system. Here, we will review the current knowledge of evading and immunosuppressive mechanisms elicited by Brucella species to persist stealthily in their hosts, such as those triggered by their LPS and cyclic ß-1,2-d-glucan or involved in neutrophil and monocyte avoidance, antigen presentation impairment, the modulation of T cell responses and immunometabolism. Attractive strategies exploited by other successful chronic pathogenic bacteria, including Mycobacteria, Salmonella, and Chlamydia, will be also discussed, with a special emphasis on the mechanisms operating in brucellosis, such as granuloma formation, pyroptosis, and manipulation of type I and III IFNs, B cells, innate lymphoid cells, and host lipids. A better understanding of these stratagems is essential to fighting bacterial chronic infections and designing innovative treatments and vaccines.

Brucella: Reservoirs and Niches in Animals and Humans.

Brucella is an intracellular bacterium that causes abortion, reproduction failure in livestock and leads to a debilitating flu-like illness with serious chronic complications if untreated in humans. As a successful intracellular pathogen, Brucella has developed strategies to avoid recognition by the immune system of the host and promote its survival and replication. In vivo, Brucellae reside mostly within phagocytes and other cells including trophoblasts, where they establish a preferred replicative niche inside the endoplasmic reticulum. This process is central as it gives Brucella the ability to maintain replicating-surviving cycles for long periods of time, even at low bacterial numbers, in its cellular niches. In this review, we propose that Brucella takes advantage of the environment provided by the cellular niches in which it resides to generate reservoirs and disseminate to other organs. We will discuss how the favored cellular niches for Brucella infection in the host give rise to anatomical reservoirs that may lead to chronic infections or persistence in asymptomatic subjects, and which may be considered as a threat for further contamination. A special emphasis will be put on bone marrow, lymph nodes, reproductive and for the first time adipose tissues, as well as wildlife reservoirs.

Nfkbie-deficiency leads to increased susceptibility to develop B-cell lymphoproliferative disorders in aged mice.

Aberrant NF-κB activation is a hallmark of most B-cell malignancies. Recurrent inactivating somatic mutations in the NFKBIE gene, which encodes IκBε, an inhibitor of NF-κB-inducible activity, are reported in several B-cell malignancies with highest frequencies in chronic lymphocytic leukemia and primary mediastinal B-cell lymphoma, and account for a fraction of NF-κB pathway activation. The impact of NFKBIE deficiency on B-cell development and function remains, however, largely unknown. Here, we show that Nfkbie-deficient mice exhibit an amplification of marginal zone B cells and an expansion of B1 B-cell subsets. In germinal center (GC)-dependent immune response, Nfkbie deficiency triggers expansion of GC B-cells through increasing cell proliferation in a B-cell autonomous manner. We also show that Nfkbie deficiency results in hyperproliferation of a B1 B-cell subset and leads to increased NF-κB activation in these cells upon Toll-like receptor stimulation. Nfkbie deficiency cooperates with mutant MYD88 signaling and enhances B-cell proliferation in vitro. In aged mice, Nfkbie absence drives the development of an oligoclonal indolent B-cell lymphoproliferative disorders, resembling monoclonal B-cell lymphocytosis. Collectively, these findings shed light on an essential role of IκBε in finely tuning B-cell development and function.

Omp25-dependent engagement of SLAMF1 by Brucella abortus in dendritic cells limits acute inflammation and favours bacterial persistence in vivo.

The strategies by which intracellular pathogenic bacteria manipulate innate immunity to establish chronicity are poorly understood. Here, we show that Brucella abortus outer membrane protein Omp25 specifically binds the immune cell receptor SLAMF1 in vitro. The Omp25-dependent engagement of SLAMF1 by B. abortus limits NF-κB translocation in dendritic cells (DCs) with no impact on Brucella intracellular trafficking and replication. This in turn decreases pro-inflammatory cytokine secretion and impairs DC activation. The Omp25-SLAMF1 axis also dampens the immune response without affecting bacterial replication in vivo during the acute phase of Brucella infection in a mouse model. In contrast, at the chronic stage of infection, the Omp25/SLAMF1 engagement is essential for Brucella persistence. Interaction of a specific bacterial protein with an immune cell receptor expressed on the DC surface at the acute stage of infection is thus a powerful mechanism to support microbe settling in its replicative niche and progression to chronicity.

Immunomodulatory properties of Brucella melitensis lipopolysaccharide determinants on mouse dendritic cells in vitro and in vivo.

The lipopolysaccharide (LPS) is a major virulence factor of Brucella, a facultative intracellular pathogenic Gram-negative bacterium. Brucella LPS exhibits a low toxicity and its atypical structure was postulated to delay the host immune response, favouring the establishment of chronic disease. Here we carried out an in-depth in vitro and in vivo characterisation of the immunomodulatory effects of Brucella LPS on different dendritic cell (DC) subpopulations. By using LPSs from bacteria that share some of Brucella LPS structural features, we demonstrated that the core component of B. melitensis wild-type (Bm-wt) LPS accounts for the low activation potential of Brucella LPS in mouse GM-CSF-derived (GM-) DCs. Contrary to the accepted dogma considering Brucella LPS a poor TLR4 agonist and DC activator, Bm-wt LPS selectively induced expression of surface activation markers and cytokine secretion from Flt3-Ligand-derived (FL-) DCs in a TLR4-dependent manner. It also primed in vitro T cell proliferation by FL-DCs. In contrast, modified LPS with a defective core purified from Brucella carrying a mutated wadC gene (Bm-wadC), efficiently potentiated mouse and human DC activation and T cell proliferation in vitro. In vivo, Bm-wt LPS promoted scant activation of splenic DC subsets and limited recruitment of monocyte- DC like cells in the spleen, conversely to Bm-wadC LPS. Bm-wadC live bacteria drove high cytokine secretion levels in sera of infected mice. Altogether, these results illustrate the immunomodulatory properties of Brucella LPS and the enhanced DC activation ability of the wadC mutation with potential for vaccine development targeting Brucella core LPS structure.

Dual ligand/receptor interactions activate urothelial defenses against uropathogenic E. coli.

During urinary tract infection (UTI), the second most common bacterial infection, dynamic interactions take place between uropathogenic E. coli (UPEC) and host urothelial cells. While significant strides have been made in the identification of the virulence factors of UPEC, our understanding of how the urothelial cells mobilize innate defenses against the invading UPEC remains rudimentary. Here we show that mouse urothelium responds to the adhesion of type 1-fimbriated UPEC by rapidly activating the canonical NF-κB selectively in terminally differentiated, superficial (umbrella) cells. This activation depends on a dual ligand/receptor system, one between FimH adhesin and uroplakin Ia and another between lipopolysaccharide and Toll-like receptor 4. When activated, all the nuclei (up to 11) of a multinucleated umbrella cell are affected, leading to significant amplification of proinflammatory signals. Intermediate and basal cells of the urothelium undergo NF-κB activation only if the umbrella cells are detached or if the UPEC persistently express type 1-fimbriae. Inhibition of NF-κB prevents the urothelium from clearing the intracellular bacterial communities, leading to prolonged bladder colonization by UPEC. Based on these data, we propose a model of dual ligand/receptor system in innate urothelial defenses against UPEC.

I kappa B kinase alpha (IKKα) activity is required for functional maturation of dendritic cells and acquired immunity to infection.

Dendritic cells (DC) are required for priming antigen-specific T cells and acquired immunity to many important human pathogens, including Mycobacteriuim tuberculosis (TB) and influenza. However, inappropriate priming of auto-reactive T cells is linked with autoimmune disease. Understanding the molecular mechanisms that regulate the priming and activation of naïve T cells is critical for development of new improved vaccines and understanding the pathogenesis of autoimmune diseases. The serine/threonine kinase IKKα (CHUK) has previously been shown to have anti-inflammatory activity and inhibit innate immunity. Here, we show that IKKα is required in DC for priming antigen-specific T cells and acquired immunity to the human pathogen Listeria monocytogenes. We describe a new role for IKKα in regulation of IRF3 activity and the functional maturation of DC. This presents a unique role for IKKα in dampening inflammation while simultaneously promoting adaptive immunity that could have important implications for the development of new vaccine adjuvants and treatment of autoimmune diseases.

Skeletal muscle stem cells adopt a dormant cell state post mortem and retain regenerative capacity.

The accessibility to stem cells from healthy or diseased individuals, and the maintenance of their potency are challenging issues for stem cell biology. Here we report the isolation of viable and functional skeletal myogenic cells from humans up to 17 days, and mice up to 14 days post mortem, much longer beyond previous reports. Muscle stem cells are enriched in post mortem tissue, suggesting a selective survival advantage compared with other cell types. Transplantation of mouse muscle and haematopoietic stem cells regenerates tissues robustly. Cellular quiescence contributes to this cell viability where cells adopt a reversible dormant state characterized by reduced metabolic activity, a prolonged lag phase before the first cell division, elevated levels of reactive oxygen species and a transcriptional status less primed for commitment. Finally, severe hypoxia, or anoxia is critical for maintaining stem cell viability and regenerative capacity. Thus, these cells provide a useful resource for studying stem cell biology.

Fungal-induced cell cycle impairment, chromosome instability and apoptosis via differential activation of NF-κB.

Microbial pathogens have developed efficient strategies to compromise host immune responses. Cryptococcus neoformans is a facultative intracellular pathogen, recognised as the most common cause of systemic fungal infections leading to severe meningoencephalitis, mainly in immunocompromised patients. This yeast is characterized by a polysaccharide capsule, which inhibits its phagocytosis. Whereas phagocytosis escape and macrophage intracellular survival have been intensively studied, extracellular survival of this yeast and restraint of host innate immune response are still poorly understood. In this study, we have investigated whether C. neoformans affected macrophage cell viability and whether NF-κB (nuclear factor-κB), a key regulator of cell growth, apoptosis and inflammation, was involved. Using wild-type (WT) as well as mutant strains of C. neoformans for the pathogen side, and WT and mutant cell lines with altered NF-κB activity or signalling as well as primary macrophages for the host side, we show that C. neoformans manipulated NF-κB-mediated signalling in a unique way to regulate macrophage cell fate and viability. On the one hand, serotype A strains reduced macrophage proliferation in a capsule-independent fashion. This growth decrease, which required a critical dosage of NF-κB activity, was caused by cell cycle disruption and aneuploidy, relying on fungal-induced modification of expression of several cell cycle checkpoint regulators in S and G2/M phases. On the other hand, C. neoformans infection induced macrophage apoptosis in a capsule-dependent manner with a differential requirement of the classical and alternative NF-κB signalling pathways, the latter one being essential. Together, these findings shed new light on fungal strategies to subvert host response through uncoupling of NF-κB activity in pathogen-controlled apoptosis and impairment of cell cycle progression. They also provide the first demonstration of induction of aneuploidy by a fungal pathogen, which may have wider implications for human health as aneuploidy is proposed to promote tumourigenesis.

Regrowing the adult brain: NF-κB controls functional circuit formation and tissue homeostasis in the dentate gyrus.

Cognitive decline during aging is correlated with a continuous loss of cells within the brain and especially within the hippocampus, which could be regenerated by adult neurogenesis. Here we show that genetic ablation of NF-κB resulted in severe defects in the neurogenic region (dentate gyrus) of the hippocampus. Despite increased stem cell proliferation, axogenesis, synaptogenesis and neuroprotection were hampered, leading to disruption of the mossy fiber pathway and to atrophy of the dentate gyrus during aging. Here, NF-κB controls the transcription of FOXO1 and PKA, regulating axogenesis. Structural defects culminated in behavioral impairments in pattern separation. Re-activation of NF-κB resulted in integration of newborn neurons, finally to regeneration of the dentate gyrus, accompanied by a complete recovery of structural and behavioral defects. These data identify NF-κB as a crucial regulator of dentate gyrus tissue homeostasis suggesting NF-κB to be a therapeutic target for treating cognitive and mood disorders.

Inhibitor of kappa B epsilon (IκBε) is a non-redundant regulator of c-Rel-dependent gene expression in murine T and B cells.

Inhibitors of kappa B (IκBs) -α, -ß and -ε effect selective regulation of specific nuclear factor of kappa B (NF-κB) dimers according to cell lineage, differentiation state or stimulus, in a manner that is not yet precisely defined. Lymphocyte antigen receptor ligation leads to degradation of all three IκBs but activation only of subsets of NF-κB-dependent genes, including those regulated by c-Rel, such as anti-apoptotic CD40 and BAFF-R on B cells, and interleukin-2 (IL-2) in T cells. We report that pre-culture of a mouse T cell line with tumour necrosis factor-α (TNF) inhibits IL-2 gene expression at the level of transcription through suppressive effects on NF-κB, AP-1 and NFAT transcription factor expression and function. Selective upregulation of IκBε and suppressed nuclear translocation of c-Rel were very marked in TNF-treated, compared to control cells, whether activated via T cell receptor (TCR) pathway or TNF receptor. IκBε associated with newly synthesised c-Rel in activated cells and, in contrast to IκBα and -ß, showed enhanced association with p65/c-Rel in TNF-treated cells relative to controls. Studies in IκBε-deficient mice revealed that basal nuclear expression and nuclear translocation of c-Rel at early time-points of receptor ligation were higher in IκBε-/- T and B cells, compared to wild-type. IκBε-/- mice exhibited increased lymph node cellularity and enhanced basal thymidine incorporation by lymphoid cells ex vivo. IκBε-/- T cell blasts were primed for IL-2 expression, relative to wild-type. IκBε-/- splenic B cells showed enhanced survival ex vivo, compared to wild-type, and survival correlated with basal expression of CD40 and induced expression of CD40 and BAFF-R. Enhanced basal nuclear translocation of c-Rel, and upregulation of BAFF-R and CD40 occurred despite increased IκBα expression in IκBε-/- B cells. The data imply that regulation of these c-Rel-dependent lymphoid responses is a non-redundant function of IκBε.

H. pylori-induced promoter hypermethylation downregulates USF1 and USF2 transcription factor gene expression.

Helicobacter pylori infection is associated with the development of gastric adenocarcinoma. Upstream stimulatory factors USF1 and USF2 regulate the transcription of genes related to immune response, cell cycle and cell proliferation. A decrease in their expression is observed in human gastric epithelial cells infected with H. pylori, associated to a lower binding to their DNA E-box recognition site as shown by electrophoretic mobility shift assay. DNA methylation leads to gene silencing. The treatment of cells with 5'-azacytidine, an inhibitor of DNA methylation, restored the USF1 and USF2 gene expression in the presence of infection. Using promoter PCR methylation assay, a DNA hypermethylation was shown in the promoter region of USF1 and USF2 genes, in infected cells. The inhibition of USF1 and USF2 expression by H. pylori and the DNA hypermethylation in their gene promoter region was confirmed in gastric tissues isolated from 12 to 18 months infected mice. Our study demonstrated the involvement of USF1 and USF2 as molecular targets of H. pylori and the key role of DNA methylation in their regulation. These mechanisms occurred in the context of metaplastic lesions, suggesting that alteration of USF1 and USF2 levels could participate in the promotion of neoplastic process during H. pylori infection.

Anthrax lethal toxin down-regulates type-IIA secreted phospholipase A(2) expression through MAPK/NF-kappaB inactivation.

Bacillus anthracis, the etiological agent of anthrax, produces lethal toxin (LT) that displays a metallo-proteolytic activity toward the N-terminus of the MAPK-kinases. We have previously shown that secreted type-IIA phospholipase A(2) (sPLA(2)-IIA) exhibits potent anthracidal activity. In vitro expression of sPLA(2)-IIA in guinea pig alveolar macrophages (AMs), the major source of this enzyme in lung tissues, is inhibited by LT. Here, we examined the mechanisms involved in sPLA(2)-IIA inhibition by LT. We first showed that chemical inhibitors of p38 and ERK MAPKs reduced sPLA(2)-IIA expression in AMs indicating that these kinases play a role in sPLA(2)-IIA expression. LT inhibited IL-1beta-induced p38 phosphorylation as well as sPLA(2)-IIA promoter activity in CHO cells. Inhibition of sPLA(2)-IIA promoter activity was mimicked by co-transfection with dominant negative construct of p38 (DN-p38) and reversed by the active form of p38-MAPK (AC-p38). Both LT and DN-p38 decreased IL-1beta-induced NF-kappaB luciferase activity. This contrasted with the effect of AC-p38, which enhanced this activity. However, neither LT nor specific p-38 inhibitor interfered with LPS-induced IkappaBalpha degradation or NF-kappaB nuclear translocation in AMs. Subcutaneous administration of LT to guinea pig before LPS challenge reduced sPLA(2)-IIA levels in broncho-alveolar lavages and ears. We conclude that sPLA(2)-IIA expression is induced via a sequential MAPK-NF-kappaB activation and that LT inhibits this expression likely by interfering with the transactivation of NF-kappaB in the nucleus. This inhibition, which is operating both in vitro and in vivo, may represent a mechanism by which B. anthracis subvert host defense.

NF-kappaB activation in endothelial cells is critical for the activity of angiostatic agents.

In tumor cells, the transcription factor NF-kappaB has been described to be antiapoptotic and proproliferative and involved in the production of angiogenic factors such as vascular endothelial growth factor. From these data, a protumorigenic role of NF-kappaB has emerged. Here, we examined in endothelial cells whether NF-kappaB signaling pathway is involved in mediating the angiostatic properties of angiogenesis inhibitors. The current report describes that biochemically unrelated agents with direct angiostatic effect induced NF-kappaB activation in endothelial cells. Our data showed that endostatin, anginex, angiostatin, and the 16-kDa N-terminal fragment of human prolactin induced NF-kappaB activation in endothelial cells in both cultured human endothelial cells and in vivo in a mouse tumor model. It was also found that NF-kappaB activity was required for the angiostatic activity, because inhibition of NF-kappaB in endothelial cells impaired the ability of angiostatic agents to block sprouting of endothelial cells and to overcome endothelial cell anergy. Therefore, activation of NF-kappaB in endothelial cells can result in an unexpected antitumor outcome. Based on these data, the current approach of systemic treatment with NF-kappaB inhibitors may therefore be revisited because NF-kappaB activation specifically targeted to endothelial cells might represent an efficient strategy for the treatment of cancer.

Timed NF-kappaB inhibition in skin reveals dual independent effects on development of HED/EDA and chronic inflammation.

We have shown earlier that inhibiting NF-kappaB activity in murine basal keratinocytes leads to hyperproliferation, inflammation, and cancer in a tumor necrosis factor receptor 1 (TNFR1)-dependent manner. We report here the outcomes of NF-kappaB abrogation at different stages of epidermal morphogenesis using a conditional IkappaBalpha transgenic mouse model. We find that blocking NF-kappaB during embryogenesis mimics the epidermal and glandular defects seen in the human disease hypohidrotic/anhidrotic ectodermal dysplasia (HED/EDA), independently of the inflammatory phenotype and TNFR1. The onset of transgene expression after birth correlates with nuclear exclusion of the NF-kappaB p50 subunit, hyperplasia, and development of a chronic inflammation initiated and dominated by macrophages. In this model, macrophages are important producers of the vascular endothelial growth factor A (VEGFA), whose inhibition attenuates the excessive angiogenesis otherwise observed. The inflammatory reaction requires the continuous suppression of NF-kappaB in keratinocytes, indicating that an immune cell attractant(s) is directly induced in response to NF-kappaB inhibition. As TNFalpha upregulation is a late event in this model, good candidates for such chemoattraction are the monocyte chemotactic proteins 1, 2, and 3 (MCP-1-2-3), which are upregulated in the epidermal compartment concomitantly with the onset of NF-kappaB inhibition.

Wild-derived mouse strains, a valuable model to study B cell responses.

In the present report, we revisited the B cell responsiveness of 7 wild-derived mouse strains to various toll-like receptor ligands (TLR-L). We found that 2 of them, namely PWK and STF presented profound defects in B cell proliferative responses to most of the TLR-L. Yet, their macrophage responses were largely unaffected, suggesting that regulation of TLR pathways are distinct in B cells and macrophages. We also showed that, anti-CD40 mAbs rescued the low proliferative responses to CpG in both PWK and STF B cells. In the other hand, CpG synergized with LPS to induce high levels of proliferation in STF B cells, which did not respond to LPS alone. Cytokine or immunoglobulin (Ig) productions, in vitro, were less impaired than the proliferative responses to LPS or CpG alone. In STF B cells, both ERK, P38 and JNK pathways were affected following in vitro TLR4 or TLR9 signaling. Moreover, while the basal levels of Ig secreting cells and of serum Igs were similar to that of control mice, antibody responses to both TI and TD antigens were severely affected, mainly in STF mice. Our findings therefore highlight the relevance of wild-derived mouse strains and TLR-L to study B cell physiology.

Toll-like receptor 4 in lymphatic endothelial cells contributes to LPS-induced lymphangiogenesis by chemotactic recruitment of macrophages.

The lymphatic vessel is a major conduit for immune cell transport; however, little is known about how lymphatic vessels regulate immune cell trafficking and how lymphatic vessels themselves respond to inflammation. Toll-like receptor 4 (TLR4) plays a central role in lipopolysaccharide (LPS)-induced inflammation, but the role of TLR4 in lymphatic endothelial cells (LECs) is poorly understood. Here, we found that LECs express high amounts of TLR4 in the intracellular region, and that the TLR4 of LECs is the main mediator of nuclear factor-kappaB (NF-kappaB) activation by LPS. LPS-TLR4 signaling in LECs resulted in the production of various chemokines for chemotaxis of macrophage. In addition, TLR4 in LECs actively contributed to the recruitment of macrophages to the draining lymphatic vessel. Furthermore, the macrophages that infiltrated into the lymphatic vessel induced lymphangiogenesis by secreting lymphangiogenic growth factors. These phenomena were largely attenuated not only in the mice defective in TLR4 signaling but also in the chimeric mice defective in TLR4 signaling that were recipients for bone marrow transplantation from normal TLR4-signaling mice. In conclusion, TLR4 in LECs plays an essential role in LPS-induced inflammatory lymphangiogenesis by chemotactic recruitment of macrophages.

NF-kappaB activation during acute Helicobacter pylori infection in mice.

Nuclear factor kappaB (NF-kappaB) plays a key regulatory role in host cell responses to Helicobacter pylori infection in humans. Although mice are routinely used as a model to study H. pylori pathogenesis, the role of NF-kappaB in murine cell responses to helicobacters has not been studied in detail. We thus investigated the abilities of different Helicobacter isolates to induce NF-kappaB-dependent responses in murine gastric epithelial cells (GECs) and in transgenic mice harboring an NF-kappaB-responsive lacZ reporter gene. H. pylori and Helicobacter felis strains up-regulated the synthesis in mouse GECs of the NF-kappaB-dependent chemokines KC (CXCL1) and MIP-2 (CXCL2). These responses were cag pathogenicity island (cagPAI) independent and could be abolished by pretreatment with a pharmacological inhibitor of NF-kappaB. Consistent with the in vitro data, experimental Helicobacter infection of transgenic mice resulted in increased numbers of GECs with nuclear beta-galactosidase activity, which is indicative of specific NF-kappaB activation. The numbers of beta-galactosidase-positive cells in mice were significantly increased at day 1 postinoculation with wild-type H. pylori strains harboring or not harboring a functional cagPAI, compared to naive animals (P = 0.007 and P = 0.04, respectively). Strikingly, however, no differences were observed in the levels of gastric NF-kappaB activation at day 1 postinoculation with H. felis or at day 30 or 135 postinoculation with H. pylori. This work demonstrates for the first time the induction of NF-kappaB activation within gastric mucosal cells during acute H. pylori infection. Furthermore, the data suggest that helicobacters may be able to regulate NF-kappaB signaling during chronic infection.

Lymphatic vessel density and function in experimental bladder cancer.

BACKGROUND: The lymphatics form a second circulatory system that drains the extracellular fluid and proteins from the tumor microenvironment, and provides an exclusive environment in which immune cells interact and respond to foreign antigen. Both cancer and inflammation are known to induce lymphangiogenesis. However, little is known about bladder lymphatic vessels and their involvement in cancer formation and progression. METHODS: A double transgenic mouse model was generated by crossing a bladder cancer-induced transgenic, in which SV40 large T antigen was under the control of uroplakin II promoter, with another transgenic mouse harboring a lacZ reporter gene under the control of an NF-kappaB-responsive promoter (kappaB-lacZ) exhibiting constitutive activity of beta-galactosidase in lymphatic endothelial cells. In this new mouse model (SV40-lacZ), we examined the lymphatic vessel density (LVD) and function (LVF) during bladder cancer progression. LVD was performed in bladder whole mounts and cross-sections by fluorescent immunohistochemistry (IHC) using LYVE-1 antibody. LVF was assessed by real-time in vivo imaging techniques using a contrast agent (biotin-BSA-Gd-DTPA-Cy5.5; Gd-Cy5.5) suitable for both magnetic resonance imaging (MRI) and near infrared fluorescence (NIRF). In addition, IHC of Cy5.5 was used for time-course analysis of co-localization of Gd-Cy5.5 with LYVE-1-positive lymphatics and CD31-positive blood vessels. RESULTS: SV40-lacZ mice develop bladder cancer and permitted visualization of lymphatics. A significant increase in LVD was found concomitantly with bladder cancer progression. Double labeling of the bladder cross-sections with LYVE-1 and Ki-67 antibodies indicated cancer-induced lymphangiogenesis. MRI detected mouse bladder cancer, as early as 4 months, and permitted to follow tumor sizes during cancer progression. Using Gd-Cy5.5 as a contrast agent for MRI-guided lymphangiography, we determined a possible reduction of lymphatic flow within the tumoral area. In addition, NIRF studies of Gd-Cy5.5 confirmed its temporal distribution between CD31-positive blood vessels and LYVE-1 positive lymphatic vessels. CONCLUSION: SV40-lacZ mice permit the visualization of lymphatics during bladder cancer progression. Gd-Cy5.5, as a double contrast agent for NIRF and MRI, permits to quantify delivery, transport rates, and volumes of macromolecular fluid flow through the interstitial-lymphatic continuum. Our results open the path for the study of lymphatic activity in vivo and in real time, and support the role of lymphangiogenesis during bladder cancer progression.

Bladder inflammatory transcriptome in response to tachykinins: neurokinin 1 receptor-dependent genes and transcription regulatory elements.

BACKGROUND: Tachykinins (TK), such as substance P, and their neurokinin receptors which are ubiquitously expressed in the human urinary tract, represent an endogenous system regulating bladder inflammatory, immune responses, and visceral hypersensitivity. Increasing evidence correlates alterations in the TK system with urinary tract diseases such as neurogenic bladders, outflow obstruction, idiopathic detrusor instability, and interstitial cystitis. However, despite promising effects in animal models, there seems to be no published clinical study showing that NK-receptor antagonists are an effective treatment of pain in general or urinary tract disorders, such as detrusor overactivity. In order to search for therapeutic targets that could block the tachykinin system, we set forth to determine the regulatory network downstream of NK1 receptor activation. First, NK1R-dependent transcripts were determined and used to query known databases for their respective transcription regulatory elements (TREs). METHODS: An expression analysis was performed using urinary bladders isolated from sensitized wild type (WT) and NK1R-/- mice that were stimulated with saline, LPS, or antigen to provoke inflammation. Based on cDNA array results, NK1R-dependent genes were selected. PAINT software was used to query TRANSFAC database and to retrieve upstream TREs that were confirmed by electrophoretic mobility shift assays. RESULTS: The regulatory network of TREs driving NK1R-dependent genes presented cRel in a central position driving 22% of all genes, followed by AP-1, NF-kappaB, v-Myb, CRE-BP1/c-Jun, USF, Pax-6, Efr-1, Egr-3, and AREB6. A comparison between NK1R-dependent and NK1R-independent genes revealed Nkx-2.5 as a unique discriminator. In the presence of NK1R, Nkx2-5 _01 was significantly correlated with 36 transcripts which included several candidates for mediating bladder development (FGF) and inflammation (PAR-3, IL-1R, IL-6, alpha-NGF, TSP2). In the absence of NK1R, the matrix Nkx2-5_02 had a predominant participation driving 8 transcripts, which includes those involved in cancer (EYA1, Trail, HSF1, and ELK-1), smooth-to-skeletal muscle trans-differentiation, and Z01, a tight-junction protein, expression. Electrophoretic mobility shift assays confirmed that, in the mouse urinary bladder, activation of NK1R by substance P (SP) induces both NKx-2.5 and NF-kappaB translocations. CONCLUSION: This is the first report describing a role for Nkx2.5 in the urinary tract. As Nkx2.5 is the unique discriminator of NK1R-modulated inflammation, it can be imagined that in the near future, new based therapies selective for controlling Nkx2.5 activity in the urinary tract may be used in the treatment in a number of bladder disorders.