A Pbx1-dependent genetic and transcriptional network regulates spleen ontogeny

The genetic control of cell fate specification, morphogenesis and expansion of the spleen, a crucial lymphoid organ, is poorly understood. Recent studies of mutant mice implicate various transcription factors in spleen development, but the hierarchical relationships between these factors have not been explored. In this report, we establish a genetic network that regulates spleen ontogeny, by analyzing asplenic mice mutant for the transcription factors Pbx1, Hox11 (Tlx1), Nkx3.2 (Bapx1) and Pod1 (capsulin, Tcf21). We show that Hox11 and Nkx2.5, among the earliest known markers for splenic progenitor cells, are absent in the splenic anlage of Pbx1 homozygous mutant (-/-) embryos, implicating the TALE homeoprotein Pbx1 in splenic cell specification. Pbx1 and Hox11 genetically interact in spleen formation and loss of either is associated with a similar reduction of progenitor cell proliferation and failed expansion of the splenic anlage. Chromatin immunoprecipitation assays show that Pbx1 binds to the Hox11 promoter in spleen mesenchymal cells, which co-express Pbx1 and Hox11. Furthermore, Hox11 binds its own promoter in vivo and acts synergistically with TALE proteins to activate transcription, supporting its role in an auto-regulatory circuit. These studies establish a Pbx1-Hox11-dependent genetic and transcriptional pathway in spleen ontogeny. Additionally, we demonstrate that while Nkx3.2 and Pod1 control spleen development via separate pathways, Pbx1 genetically regulates key players in both pathways, and thus emerges as a central hierarchical co-regulator in spleen genesis

Impaired testicular signaling of vitamin A and vitamin K contributes to the aberrant composition of the extracellular matrix in idiopathic germ cell aplasia

Objective: To study pathogenic features of the somatic testicular microenvironment associated with idiopathic germ cell aplasia. Design: Cross-sectional study. Setting: Tertiary referral center for reproductive medicine. Patient(s): Testicular specimens from men with idiopathic nonobstructive azoospermia (iNOA) prospectively submitted to microdissection testicular sperm extraction. Of 20 specimens used for histology, 10 were also available for proteomic analysis. Primary Sertoli cells with normal karyotype and phenotype were also used. Intervention(s): Patients with iNOA were dichotomized according to a positive versus negative sperm retrieval at microdissection testicular sperm extraction, and on the isolated extracellular matrix (ECM) the proteomic analysis was performed. Main Outcome Measure(s): Proteomic analysis of the ECM from testicular specimens with positive versus negative sperm retrieval. Gene ontology enrichment was used to identify upstream regulators based on the 11 deregulated ECM proteins, which were validated by immunohistochemistry and quantitative polymerase chain reaction. Continuous variables were expressed as medians and interquartile range. Result(s): Germ cell aplasia was characterized by an increased signaling of the retinoic acid in Sertoli cells and associated with decreased expression of the basal membrane markers nidogen-2 and heparan sulfate proteoglycan-2. Decreased levels of the interstitial matrisome-associated factor IX and its regulator VKORC1 were, instead, coupled with decreased signaling of vitamin K in Leydig cells. An altered expression of a further eight ECM proteins was also found, including laminin-4 and laminin-5. Peripheral levels of the two vitamins were within the reference range in the two cohorts of iNOA men. Conclusion(s): We identified the pathogenetic signature of the somatic human testicular microenvironment, providing two vitamin-related mechanistic insights related to the molecular determinants of the idiopathic germ cell aplasia

Intra-parenchymal renal resistive index variation (IRRIV) describes renal functional reserve (RFR): Pilot study in healthy volunteers

An increase of glomerular filtration rate after protein load represents renal functional reserve (RFR) and is due to afferent arteriolar vasodilation. Lack of RFR may be a risk factor for acute kidney injury (AKI), but is cumbersome to measure. We sought to develop a non-invasive, bedside method that would indirectly measure RFR. Mechanical abdominal pressure, through compression of renal vessels, decreases blood flow and activates the auto-regulatory mechanism which can be measured by a fall in renal resistive index (RRI). The study aims at elucidating the relationship between intra-parenchymal renal resistive index variation (IRRIV) during abdominal pressure and RFR. In healthy volunteers, pressure was applied by a weight on the abdomen (fluid-bag 10% of subject's body weight) while RFR was measured through a protein loading test. We recorded RRI in an interlobular artery after application of pressure using ultrasound. The maximum percentage reduction of RRI from baseline was compared in the same subject to RFR. We enrolled 14 male and 16 female subjects (mean age 38 ± 14 years). Mean creatinine clearance was 106.2 ± 16.4 ml/min/1.73 m2. RFR ranged between -1.9 and 59.7 with a mean value of 28.9 ± 13.1 ml/min/1.73 m2. Mean baseline RRI was 0.61 ± 0.05, compared to 0.49 ± 0.06 during abdominal pressure; IRRIV was 19.6 ± 6.7%, ranging between 3.1% and 29.2%. Pearson's coefficient between RFR and IRRIV was 74.16% (p < 0.001). Our data show the correlation between IRRIV and RFR. Our results can lead to the development of a "stress test" for a rapid screen of RFR to establish renal susceptibility to different exposures and the consequent risk for AKI

Determinants of postnatal spleen tissue regeneration and organogenesis

Abstract The spleen is an organ that filters the blood and is responsible for generating blood-borne immune responses. It is also an organ with a remarkable capacity to regenerate. Techniques for splenic auto-transplantation have emerged to take advantage of this characteristic and rebuild spleen tissue in individuals undergoing splenectomy. While this procedure has been performed for decades, the underlying mechanisms controlling spleen regeneration have remained elusive. Insights into secondary lymphoid organogenesis and the roles of stromal organiser cells and lymphotoxin signalling in lymph node development have helped reveal similar requirements for spleen regeneration. These factors are now considered in the regulation of embryonic and postnatal spleen formation, and in the establishment of mature white pulp and marginal zone compartments which are essential for spleen-mediated immunity. A greater understanding of the cellular and molecular mechanisms which control spleen development will assist in the design of more precise and efficient tissue grafting methods for spleen regeneration on demand. Regeneration of organs which harbour functional white pulp tissue will also offer novel opportunities for effective immunotherapy against cancer as well as infectious diseases

Impaired Spleen Formation Perturbs Morphogenesis of the Gastric Lobe of the Pancreas

Despite the extensive use of the mouse as a model for studies of pancreas development and disease, the development of the gastric pancreatic lobe has been largely overlooked. In this study we use optical projection tomography to provide a detailed three-dimensional and quantitative description of pancreatic growth dynamics in the mouse. Hereby, we describe the epithelial and mesenchymal events leading to the formation of the gastric lobe of the pancreas. We show that this structure forms by perpendicular growth from the dorsal pancreatic epithelium into a distinct lateral domain of the dorsal pancreatic mesenchyme. Our data support a role for spleen organogenesis in the establishment of this mesenchymal domain and in mice displaying perturbed spleen development, including Dh +/−, Bapx1−/− and Sox11−/−, gastric lobe development is disturbed. We further show that the expression profile of markers for multipotent progenitors is delayed in the gastric lobe as compared to the splenic and duodenal pancreatic lobes. Altogether, this study provides new information regarding the developmental dynamics underlying the formation of the gastric lobe of the pancreas and recognizes lobular heterogeneities regarding the time course of pancreatic cellular differentiation. Collectively, these data are likely to constitute important elements in future interpretations of the developing and/or diseased pancreas

Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses

Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients