[Models of glomerular filtration barrier : New developments]. / Modélisation de la barrière de filtration glomérulaire - Nouvelles avancées.

In this article, we present the latest innovations to generate in vitro models of the glomerular filtration barrier. There is currently a growing interest for such model systems that allow to reduce the use of animal models. Methodologies to improve their physiological relevance have taken advantage of the development of induced pluripotent stem cells and of bioengineering, particularly tissue engineering. Here, we first introduce the methods to overcome the limitations of the currently used glomerular cells based on the use of stem cells. The different approaches to obtain podocytes, the most important cells in the glomerulus, are presented. Finally, we emphasize the importance of the glomerular microenvironment in maintaining the glomerular cell phenotype, which can be achieved by co-culturing different glomerular cells, integration of biomaterials mimicking the extracellular matrix and introduction of flows with microfluidics.

TITLE: Modélisation de la barrière de filtration glomérulaire - Nouvelles avancées. ABSTRACT: Nous présentons, dans cette revue, les dernières avancées concernant la modélisation in vitro de la barrière de filtration glomérulaire. Ces systèmes, permettant de réduire l'utilisation des modèles animaux, connaissent un intérêt croissant et bénéficient du développement de nos connaissances des cellules souches et de la bioingénierie. Nous discuterons les limites des modèles cellulaires glomérulaires actuels et nous introduirons les méthodes permettant d'obtenir des cellules glomérulaires à partir des cellules souches. Enfin, nous discuterons de l'importance du microenvironnement dans le maintien du phénotype, quels que soient les systèmes utilisés tels que la co-culture, les biomatériaux ou la microfluidique.

HLA-D and PLA2R1 risk alleles associate with recurrent primary membranous nephropathy in kidney transplant recipients.

Recurrence of primary membranous nephropathy after transplantation occurs in up to 44% of patients and is driven by PLA2R antibody. Here, we asked whether genetic determinants could improve risk prediction. First, we sequenced PLA2R1 and HLA-D loci in 248 patients with primary membranous nephropathy and identified two independent single nucleotide polymorphisms (SNPs) at risk for primary membranous nephropathy at each locus. These were rs9271188 (intergenic between HLA-DRB1 and HLA-DQA1,) and rs9275086 (intergenic between HLA-DQB1 and HLA-DQA2) at the HLA-D locus along with rs6726925 and rs13018963 at the PLA2R1 locus. Then we investigated whether primary membranous nephropathy at-risk variants were associated with recurrence in a retrospective cohort of 105 donor-recipient pairs and a replication cohort of 40 pairs. Seven SNPs located between HLA-DRB1 and HLA-DQA1 in linkage disequilibrium with rs9271188, and three SNPs in the PLA2R1 region predicted recurrence when presented by the donor, but not when presented by the recipient. The two SNPs in the HLA-D region most strongly associated with recurrence (rs9271705 and rs9271550) were confirmed in the replication cohort. A genetic risk score based on the two best predictors at each locus (rs9271705, rs9271550, rs17830558, and rs3828323) identified a group of patients with high risk of recurrence. Thus, our results suggest that the graft contributes to recurrence of primary membranous nephropathy through the disease susceptibility HLA-D and PLA2R1 SNPs in an autoimmune milieu. Further studies are needed before implementation of genetic testing for these in donor selection.

zHSF1 modulates zper2 expression in zebrafish embryos.

HSF1 is a transcription factor that plays a key role in circadian resetting by temperature. We have used zebrafish embryos to decipher the roles of zHsf1, heat and light on zper2 transcription in vivo. Our results show that heat shock (HS) stimulated zper2 expression in the dark but has no cumulative effect combined with light. After light exposition, zper2 expression was 2.7 fold increased threefold in the hsf1-morphants in comparison to control embryos. Our results show that zHsf1 plays a positive role in HS-driven expression of zper2 in the dark but seems to act as an attenuator in the presence light.

Extracellular HSP110 skews macrophage polarization in colorectal cancer.

HSP110 is induced by different stresses and, through its anti-apoptotic and chaperoning properties, helps the cells to survive these adverse situations. In colon cancers, HSP110 is abnormally abundant. We have recently showed that colorectal cancer (CRC) patients with microsatellite instability (MSI) had an improved response to chemotherapy because they harbor an HSP110 inactivating mutation (HSP110DE9). In this work, we have used patients' biopsies and human CRC cells grown in vitro and in vivo (xenografts) to demonstrate that (1) HSP110 is secreted by CRC cells and that the amount of this extracellular HSP110 is strongly decreased by the expression of the mutant HSP110DE9, (2) Supernatants from CRC cells overexpressing HSP110 or purified recombinant human HSP110 (LPS-free) affect macrophage differentiation/polarization by favoring a pro-tumor, anti-inflammatory profile, (3) Conversely, inhibition of HSP110 (expression of siRNA, HSP110DE9 or immunodepletion) induced the formation of macrophages with a cytotoxic, pro-inflammatory profile. (4) Finally, this effect of extracellular HSP110 on macrophages seems to implicate TLR4. These results together with the fact that colorectal tumor biopsies with HSP110 high were infiltrated with macrophages with a pro-tumoral profile while those with HSP110 low were infiltrated with macrophages with a cytotoxic profile, suggest that the effect of extracellular HSP110 function on macrophages may also contribute to the poor outcomes associated with HSP110 expression.

TLR4/IFNγ pathways induce tumor regression via NOS II-dependent NO and ROS production in murine breast cancer models.

Toll-like receptor (TLR) 4 agonists have emerged as a new group of molecules used for cancer therapy. They have been exploited to enhance the immunogenicity of current chemotherapeutic regimens. However, their effects on cancer cells remain elusive. Here, we showed that a TLR4 agonist, namely a synthetic lipid A analog (ALA), OM-174, exhibits antitumor effects in several mammary tumor mouse models. We also showed that immune components are involved in such effects, as attested to by the failure of ALA to induce tumor regression or an increase of animal survival in mice knocked-out for interferon γ (IFNγ) or TLR4. TLR4 and IFNγ receptor (INFR2) expressed by cancer cells are involved in the antitumor efficacy of ALA since this last did not inhibit tumor growth in mice bearing a tumor but lacking TLR4 or IFNγ receptor 2 (IFNR2). Mechanistic investigations revealed that nitric oxide (NO), superoxide and peroxynitrite produced by uncoupling of inducible NO synthase (NOS II) in cancer cells are key mediators of ALA and IFNγ-mediated tumor growth inhibition. We present here a comprehensive picture of tumor cell death induction, in vivo and in vitro, by immunotherapy and for the first time the involvement of the TLR4/IFNγ/NOS II pathway in immunotherapy was investigated.

Clinical significance of T-bet, GATA-3, and Bcl-6 transcription factor expression in bladder carcinoma.

BACKGROUND: The aim of this study was to investigate the clinical significance of three immune cell-related transcription factors, T-bet, GATA-3 and Bcl-6 in bladder cancer in Tunisian patients. METHODS: Expression of T-bet, GATA-3 and Bcl-6 genes was assessed using RT-qPCR in 65 bladder cancers from patients: 32 being diagnosed as low- and medium-grade, 31 as high-grade, 25 as muscle invasive stage and 39 as non-muscle invasive stage. Gene expression was statistically correlated according to the grade, the stage, tobacco consumption, the BCG response and disease severity. RESULTS: T-bet levels in patients with high-grade bladder cancer were significantly elevated compared to patients with low- or medium-grade bladder cancer (p = 0.005). In invasive carcinoma (T2-T4), the T-bet levels were significantly higher than in superficial non-invasive bladder tumors (Tis, Ta, and T1) (p = 0.02). However, T-bet is predictive of the response to BCG. Its expression is high in good responders to BCG (p = 0.02). In contrast, the expression of GATA-3 and Bcl-6 in non-invasive carcinoma (p = 0.008 and p = 0.0003) and in patients with low- and medium-grade cancers (p = 0.001 and p < 0.0001) is significantly higher than in invasive bladder tumors and in patients with high-grade bladder carcinoma, respectively. In addition, heavy smokers, whose tumors express low levels of GATA-3 and Bcl-6, are poor responders to BCG (p = 0.01 and p = 0.03). Finally, better patient survival correlated with GATA-3 (p = 0.04) and Bcl-6 (p = 0.04) but not T-bet expression. CONCLUSIONS: Our results suggest that T-bet expression in bladder tumors could be a positive prognostic indicator of BCG therapy, even if high levels are found in high-grade and stage of the disease. However, GATA-3 and Bcl-6 expression could be considered as predictive factors for good patient survival.

The impact of tumor nitric oxide production on VEGFA expression and tumor growth in a zebrafish rat glioma xenograft model.

To investigate the effect of nitric oxide on tumor development, we established a rat tumor xenograft model in zebrafish embryos. The injected tumor cells formed masses in which nitric oxide production could be detected by the use of the cell-permeant DAF-FM-DA (diaminofluorophore 4-amino-5-methylamino-2'-7'-difluorofluorescein diacetate) and DAR-4M-AM (diaminorhodamine-4M). This method revealed that nitric oxide production could be co-localized with the tumor xenograft in 46% of the embryos. In 85% of these embryos, tumors were vascularized and blood vessels were observed on day 4 post injection. Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA. In the xenografted embryos we also found increased zebrafish vegfa expression. Glioma and zebrafish derived Vegfa and tumor Cyclin D1 expression could be down regulated by the nitric oxide scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or CPTIO. We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma.

The biofilm mode of life boosts the anti-inflammatory properties of Lactobacillus.

The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that Lactobacillus casei ATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro-inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic-fed animals, suggesting that these bacteria have anti-inflammatory properties. We treated human macrophage-differentiated monocytic THP-1 cells with supernatants of L. casei ATCC334 grown in either biofilm or planktonic cultures. TNF-α production was suppressed and the NF-κB pathway was inhibited only in the presence of supernatants from biofilms. We identified GroEL as the biofilm supernatant compound responsible, at least partially, for this anti-inflammatory effect. Gradual immunodepletion of GroEL demonstrated that the abundance of GroEL and TNF-α were inversely correlated. We confirmed that biofilm development in other Lactobacillus species affects the immune response. The biofilms supernatants of these species also contained large amounts of GroEL. Thus, our results demonstrate that the biofilm enhances the immunomodulatory effects of Lactobacillus sp. and that secreted GroEL is involved in this beneficial effect.

Extracellular HSP27 mediates angiogenesis through Toll-like receptor 3.

The heat-shock protein 27 (HSP27) is up-regulated in tumor cells and released in their microenvironment. Here, we show that extracellular HSP27 has a proangiogenic effect evidenced on chick chorioallantoic membrane. To explore this effect, we test the recombinant human protein (rhHSP27) at physiopathological doses (0.1-10 µg/ml) onto human microvascular endothelial cells (HMECs) grown as monolayers or spheroids. When added onto HMECs, rhHSP27 dose-dependently accelerates cell migration (with a peak at 5 µg/ml) and favors spheroid sprouting within 12-24 h. rhHSP27 increases VEGF gene transcription and promotes secretion of VEGF-activating VEGF receptor type 2. Increased VEGF transcription is related to NF-κB activation in 30 min. All of these effects are initiated by rhHSP27 interaction with Toll-like receptor 3 (TLR3). Such an interaction can be detected by immunoprecipitation but does not seem to be direct, as we failed to detect an interaction between rhHSP27 and monomeric TLR3 by SPR analysis. rhHSP27 is rapidly internalized with a pool of TLR3 to the endosomal compartment (within 15-30 min), which is required for NF-κB activation in a cytosolic Ca(2+)-dependent manner. The HSP27/TLR3 interaction induces NF-κB activation, leading to VEGF-mediated cell migration and angiogenesis. Such a pathway provides alternative targets for antiangiogenic cancer therapy.

SMYD3 promotes cancer invasion by epigenetic upregulation of the metalloproteinase MMP-9.

Upregulation of the matrix metalloproteinase (MMP)-9 plays a central role in tumor progression and metastasis by stimulating cell migration, tumor invasion, and angiogenesis. To gain insights into MMP-9 expression, we investigated its epigenetic control in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. Gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. Notably, we found that the H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. SMYD3 is overexpressed in many types of cancer cells, but its contributions to malignant pathophysiology are unclear. We found that overexpression of SMYD3 was sufficient to induce MMP-9 expression in transformed leukocytes and fibrosarcoma cells and that proinflammatory phorbol esters further enhanced this effect. Furthermore, SMYD3 was sufficient to increase cell migration associated with MMP-9 expression. In contrast, RNA interference-mediated knockdown of SMYD3 decreased H3K4me3 modification of the MMP-9 promoter, reduced MMP-9 expression, and reduced tumor cell proliferation. Furthermore, SMYD3 knockdown also reduced cellular invasion in a zebrafish xenograft model of cancer. Together, our results define SMYD3 as an important new regulator of MMP-9 transcription, and they provide a molecular link between SMYD3 overexpression and metastatic cancer progression.